Genes of an otitis media isolate of nontypeable haemophilus influenzae

ABSTRACT

The invention relates to the polynucleotide sequence of a nontypeable stain of  Haemophilus influenzae  (NTHi) and polypeptides encoded by the polynucleotides and uses thereof. The invention also relates to NTHi genes which are upregulated during or in response to NTHi infection of the middle ear and/or the nasopharynx.

This application is a divisional of U.S. application Ser. No. 11/917,368 filed Jan. 20, 2011, which is a U.S. National Phase filing under 35 U.S.C. §371 of International Application No. PCT/US2006/23428 filed Jun. 15, 2006, which claims priority to U.S. Provisional Application No. 60/691,214, filed Jun. 16, 2005 and U.S. patent application Ser. No. 10/795,159 filed Mar. 5, 2004, which claims priority to U.S. Provisional Application No. 60/453,134 filed Mar. 6, 2003, all of which are incorporated by reference herein in its entirety.

Part of the work during the development of this invention was made with government support from the National Institutes of Health under grant numbers R01DC03915 and R01DC005980. The U.S. Government has certain rights in the invention.

FIELD OF INVENTION

The invention relates to the polynucleotide sequence of a nontypeable strain of Haemophilus influenzae (NTHi) genome, NTHi genes contained within the genome and polypeptides encoded by the polynucleotides. The invention also relates to uses of these NTHi polynucleotides and NTHi polypeptides including vaccines and methods of treating and preventing NTHi related disorders. The invention also relates to NTHi genes which are upregulated during or in response to NTHi infection of the middle ear or nasopharynx.

BACKGROUND

Otitis media (OM) is a highly prevalent pediatric disease worldwide and is the primary cause for emergency room visits by children (Infante-Rivand and Fernandez, Epidemiol. Rev., 15: 444-465, 1993). Recent statistics indicate that 24.5 million physician office visits were made for OM in 1990, representing a greater than 200% increase over those reported in the 1980's. While rarely associated with mortality any longer, the morbidity associated with OM is significant. Hearing loss is a common problem associated with this disease, often times affecting a child's behavior, education and development of language skills (Baldwin, Am. J. Otol., 14: 601-604, 1993; Hunter et al., Ann. Otol. Rhinol. Laryngol. Suppl., 163: 59-61, 1994; Teele et al., J. Infect. Dis., 162: 685-694, 1990). The socioeconomic impact of OM is also great, with direct and indirect costs of diagnosing and managing OM exceeding $5 billion annually in the U.S. alone (Kaplan et al., Pediatr. Infect. Dis. J., 16: S9-11, 1997).

Whereas antibiotic therapy is common and the surgical placement of tympanostomy tubes has been successful in terms of draining effusions, clearing infection and relieving pain associated with the accumulation of fluids in the middle ear, the emergence of multiple antibiotic-resistant bacteria and the invasive nature associated with tube placement, has illuminated the need for more effective and accepted approaches to the management and preferably, the prevention of OM. Surgical management of chronic OM involves the insertion of tympanostomy tubes through the tympanic membrane while a child is under general anesthesia. While this procedure is commonplace (prevalence rates are ˜13%; Bright et al., Am. J. Public Health, 83(7): 1026-8, 1993) and is highly effective in terms of relieving painful symptoms by draining the middle ear of accumulated fluids, it too has met with criticism due to the invasive nature of the procedure and its incumbent risks (Berman et al., Pediatrics, 93(3):353-63, 1994; Bright et al., supra.; Cimons, ASM News, 60: 527-528; Paap, Ann. Pharmacother., 30(11): 1291-7, 1996).

Progress in vaccine development is most advanced for Streptococcus pneumoniae, the primary causative agent of acute OM (AOM), as evidenced by the recent approval and release of a seven-valent capsular-conjugate vaccine, PREVNAR® (Eskola and Kilpi, Pedriatr. Infect. Dis. J. 16: S72-78, 2000). While PREVNAR® has been highly efficacious for invasive pneumococcal disease, coverage for OM has been disappointing (6-8%) with reports of an increased number of OM cases due to serotypes not included in the vaccine (Black et al., Pedriatr. Infect. Dis J., 19: 187-195; Eskola et al., Pedriatr. Infect. Dis J., 19: S72-78, 2000; Eskola et al., N. Engl. J. Med. 344: 403-409, 2001; Snow et al., Otol. Neurotol., 23: 1-2, 2002). Less progress has been made for non-typeable Haemophilus influenzae (NTHi), the gram-negative pathogen that predominates in chronic OM with effusion (Klein, Pedriatr. Infect. Dis J., 16: S5-8, 1997; Spinola et al., J. Infect. Dis., 154: 100-109, 1986). Hampering development of effective vaccines against NTHi, is the currently incomplete understanding of the pathogenesis of NTHi-induced middle ear disease. Contributing to this delay is a lack of understanding of the dynamic interplay between microbe-expressed virulence factors and the host's immune response as the disease progresses from one of host immunological tolerance of a benign nasopharyngeal commensal, to that of an active defensive reaction to an opportunistic invader of the normally sterile middle ear space.

Although strain Rd is the exemplar organism for the current small genome sequencing rationale, and an important model organism for studying H. influenzae biology, strain Rd is a poor model for the study of pathogenicity caused by members of the genus Haemophilus. Serotype b strains of H. influenzae cause invasive diseases, for example meningitis, and nontypeable H. influenzae (NTHi) strains principally have a role in localized respiratory disease, particularly in otitis media (OM), acute sinusitis, community acquired pneumonia and have important consequences in patients with chronic obstructive pulmonary disease or cystic fibrosis (Kilpi et al., Pediatr Infect Dis J 20:654-62, 2001; Murphy, Curr Opin Infect Dis 16:129-34, 2003; Roman et al., J Clin Microbiol 42:1450-9, 2004; Sethi, Clin Microbiol Rev 14:336-63, 2001; St Geme, Vaccine 19 Suppl 1:S41-50, 2000). Strain Rd, however, is a derivative of a serotype d strain. Serotype d strains are rarely associated with disease (Daines et al., J Med Microbiol 52:277-82 2003; Heath et al., Pediatr Infect Dis J 20:300-5, 2001; Rodriguez et al., Infect Immun 71:1635-42, 2003, Skoczynska et al., J Clin Microbiol 43:938-41, 2005). Because one of the most useful sets of data in the study of an organism's biology is its genomic sequence, a number of investigations have identified and characterized genes found in H. influenzae type b strains, H. influenzae Biogroup Aegyptius strains or in nontypeable strains that are not present in strain Rd (Bergman et al., Infect Immun 71:1098-108, 2003; Chang et al., Infect Immun 68:2630-7, 2000; Erdos et al., Int J Pediatr Otorhinolaryngol 67:749-55. 2003; Li et al., Mol Microbiol 47:1101-11, 2003; McMichael & Green, Curr Opin Investig Drugs 4:953-8, 2003; Pomposiello & Demple, 2001; Smoot et al., Infect Immun 70:2694-9, 2002).

Currently there is a poor understanding of how NTHi causes OM in children. The identification of putative virulence factors necessary for induction of OM will contribute significantly to the understanding of the host-pathogen interaction and ultimately, the identification of potential vaccine candidates and targets of chemotherapy. There is a tremendous need to develop more effective and accepted approaches to the management and preferably, the prevention of otitis media. Vaccine development is a very promising and cost effective method to accomplish this goal (Giebank, Pedriatr. Infect. Dis J., 13(11): 1064-8, 1994: Karma et al., Int. J. Pedritr. Otorhinolaryngol., 32(Suppl.): S127-34, 1995).

SUMMARY OF INVENTION

The present invention provides for the identification and characterization of the genomic sequence of NTHi H. influenzae strain 86-028NP and the polypeptide sequences encoded thereby. The 3-fold analysis of the NTHi genomic sequence is set out in a series of contig sequences denoted as SEQ ID NO: 1-576, and the subsequent 8-fold analysis of the genomic sequence is set out in a series of 11 contig sequences denoted as SEQ ID NOS: 675-685. These contigs are raw data and one of skill in the art may assemble these contigs by comparing overlapping sequences to construct the complete genome of the NTHi stain 86-028NP using routine methods.

The complete and annotated genome of NTHi strain 86-028NP is set out as SEQ ID NO: 772. The open reading frames are set in Table 6 as SEQ ID NOS: 773-2593. The amino acid sequence of the resulting gene products are set out as SEQ ID NOS: 2594-4414. The genome is approximately 1.91 kb in size, slightly larger than the strain Rd genome. A number of regions of gross genome rearrangement relative to the strain Rd genome have been identified as well as a number of genes unique to strain 86-028NP.

The present invention also provides for antibodies specific for the NTHi polypeptides of the invention. Methods of detecting NTHi bacteria in a human or in sample, such as serum, sputum, ear fluid, blood, urine, lymphatic fluid and cerebrospinal fluid are contemplated. These methods include detecting NTHi polynucleotides with specific polynucleotide probes or detecting NTHi polypeptides with specific antibodies. The invention also contemplates diagnostic kits which utilize these methods of detecting NTHi bacteria.

The present invention also contemplates methods of eliciting an immune response by administering a NTHi polypeptide of the invention or a NTHi peptide thereof. These methods include administering the NTHi polypeptide or NTHi peptide as a vaccine for treatment and/or prevention of diseases caused by NTHi infection, such as OM. The following NTHi genes are upregulated during or in response to middle ear and/or nasopharynx infections; and the polypeptides encoded by these genes and peptides thereof are contemplated as possible OM vaccine candidates and/or target of chemotherapy: hisB, lppB, sapA, lolA, rbsC, purE, ribB, arcB, uxuA, dsbB, ureH, licC, HI1647, ispZ, radC, mukF, glpR, ihfB, argR, cspD, HI0094, HI1163, HI1063, HI0665, HI1292, HI1064. NTHi hisB gene is set out as nucleotide sequence SEQ ID NO: 615 and encodes the amino acid sequence set out as SEQ ID NO: 616. NTHi sapA gene is set out as nucleotide sequence SEQ ID NO: 617 and encodes the amino acid sequence set out as SEQ ID NO: 618. NTHi rbsC gene is set out as nucleotide sequence SEQ ID NO: 619 and encodes the amino acid sequence set out as SEQ ID NO: 620. NTHi purE gene is set out as nucleotide sequence SEQ ID NO: 621 and encodes the amino acid sequence set out as SEQ ID NO: 622. NTHi ribB gene is set out as nucleotide sequence SEQ ID NO: 623 and encodes the amino acid sequence set out as SEQ ID NO: 624. NTHi arcB gene is set out as nucleotide sequence SEQ ID NO: 625 and encodes the amino acid sequence set out as SEQ ID NO: 626. NTHi uxuA gene is set out as nucleotide sequence SEQ ID NO: 627 and encodes the amino acid sequence set out as SEQ ID NO: 628. NTHi dsbB gene is set out as nucleotide sequence SEQ ID NO: 629 and encodes the amino acid sequence set out as SEQ ID NO: 630. NTHi ureH gene is set out as nucleotide sequence SEQ ID NO: 631 and encodes the amino acid sequence set out as SEQ ID NO: 632. NTHi licC gene is set out as nucleotide sequence SEQ ID NO: 633 and encodes the amino acid sequence set out as SEQ ID NO: 634. NTHi HI1647 gene is set out as nucleotide sequence SEQ ID NO: 635 and encodes the amino acid sequence set out as SEQ ID NO: 636. NTHi ispZ gene is set out as nucleotide sequence SEQ ID NO: 637 and encodes the amino acid sequence set out as SEQ ID NO: 638. NTHi radC gene is set out as nucleotide sequence SEQ ID NO: 639 and encodes the amino acid sequence set out as SEQ ID NO: 640. NTHi mukF gene is set out as nucleotide sequence SEQ ID NO: 641 and encodes the amino acid sequence set out as SEQ ID NO: 642. NTHi glpR gene is set out as nucleotide sequence SEQ ID NO: 643 and encodes the amino acid sequence set out as SEQ ID NO: 644. NTHi ihfB gene is set out as nucleotide sequence SEQ ID NO: 645 and encodes the amino acid sequence set out as SEQ ID NO: 646. NTHi argR gene is set out as nucleotide sequence SEQ ID NO: 647 and encodes the amino acid sequence set out as SEQ ID NO: 648. NTHi cspD gene is set out as nucleotide sequence SEQ ID NO: 649 and encodes the amino acid sequence set out as SEQ ID NO: 650. NTHi HI1163 gene is set out as nucleotide sequence SEQ ID NO: 651 and encodes the amino acid sequence set out as SEQ ID NO: 652. NTHi HI1063 gene is set out as nucleotide sequence SEQ ID NO: 653 and encodes the amino acid sequence set out as SEQ ID NO: 654. NTHi HI0665 gene is set out as nucleotide sequence SEQ ID NO: 655 and encodes the amino acid sequence set out as SEQ ID NO: 656. NTHi HI1292 gene is set out as nucleotide sequence SEQ ID NO: 657 and encodes the amino acid sequence set out as SEQ ID NO: 658.

The novel NTHi genes included in the polynucleotide sequences presented as SEQ ID NOS: 1-576, SEQ ID NOS: 675-685 and the nucleotide sequences set out in Tables 4 and 4B are also up-regulated during infection of the middle ear and/or the nasopharynx, and therefore are contemplated to encode OM vaccine candidates and/or targets of chemotherapy. In addition, the following NTHi genes are contemplated to be virulence-associated genes and therefore are contemplated to encode possible OM vaccine candidates and/or targets of chemotherapy: HI1386, HI1462, HI1369, lay, HI1598. NTHi HI1386 gene sequence is set out as SEQ ID NO: 659 and encodes the amino acid sequence set out as SEQ ID NO: 660. NTHi HI1462 gene sequence is set out as SEQ ID NO: 661 and encodes the amino acid sequence set out as SEQ ID NO: 662. NTHi HI1369 gene sequence is set out as SEQ ID NO: 665 and encodes the amino acid sequence set out as SEQ ID NO: 666. NTHi lay gene sequence is set out as SEQ ID NO: 663 and encodes the amino acid sequence set out as SEQ ID NO: 664. NTHi HI1598 gene sequence is set out as SEQ ID NO: 669 and SEQ ID NO: 671 and encodes the amino acid sequence set out as SEQ ID NO: 670 and SEQ ID NO: 672. Additional NTHi genes associated with virulence include the polynucleotide sequences presented as SEQ ID NO: 667 and SEQ ID NO: 673.

As a method of treating or preventing NTHi infection, the present invention contemplates administering a molecule that inhibits expression or the activity of the NTHi polypeptides, which are upregulated or active during infection. In particular, the invention contemplates methods of treating or preventing NTHi infection comprising modulating NTHi protein expression by administering an antisense oligonucleotide that specifically binds to NTHi genes that are upregulated during NTHi infections, such genes include hisB, lppB, sapA, lolA, rbsC, purE, ribB, arcB, uxuA, dsbB, ureH, licC, HI1647, ispZ, radC, mukF, glpR, ihfB, argR, cspD, HI0094, HI1163, HI1063, HI0665, HI1292, HI1064. The invention also contemplates methods of treating or preventing NTHi infection comprising administering antibodies or small molecules that modulate the activity of the proteins encoded by theses genes. The novel NTHi genes included in the polynucleotide sequences presented as SEQ ID NOS: 1-576, SEQ ID NOS: 675-685 and the nucleotide sequences set out in Tables 4 and 4B are also up-regulated during infection of the middle ear and/or the nasopharynx and therefore antisense oligonucleotides that specifically bind these polynucleotide sequences are also contemplated.

Polynucleotides and Polypeptides of the Invention

The present invention provides for the sequences of the NTHi strain 86-028NP genome. The 3-fold analysis of the genomic sequence is presented as a series of contig sequences denoted herein as “contigs 1-576”. Each contig is assigned a sequence identification number that correlates with its “contig number”. Therefore, the contigs of the present invention as set out as SEQ ID NOS: 1-576. These contig polynucleotide sequences may be assembled into the complete genome sequence of the NTHi strain 86-028NP using routine methods. Upon completion of 8-fold sequence analysis of the NTHi strain 82-028NP genome, the genomic sequence was assembled into 11 contigs which are denoted herein as SEQ ID NOS: 675-685. Finally, the complete genome is presented as one nucleic acid sequence denoted herein as SEQ ID NO: 772.

The present invention provides for the NTHi polynucleotide sequences and open reading frames contained within the contigs of SEQ ID NOS: 1-576, SEQ ID NOS: 675-685, SEQ ID NOS: 773-2593, and the nucleotide sequences set out in Table 3B, Table 4B and Table 5. The present invention also provides for the polypeptide sequences encoded by the NTHi polynucleotides of the present invention such as the amino acid sequences set out in SEQ ID NOS: 2594-4414, Table 3B, Table 4B and Table 5. The invention provides for polynucleotides that hybridize under stringent conditions to (a) the complement of the nucleotides sequence of SEQ ID NOS: 1-576; SEQ ID NOS: 675-685, SEQ ID NOS: 773-2593 and the nucleotide sequences set out in Table 3B, Table 4B and Table 5 herein (b) a polynucleotide which is an allelic variant of any polynucleotides recited above; (c) a polynucleotide which encodes a species homolog of any of the proteins recited above; or (d) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of the NTHi polypeptides of the present invention.

The NTHi polynucleotides of the invention also include nucleotide sequences that are substantially equivalent to the polynucleotides recited above. Polynucleotides according to the invention can have, e.g., at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically at least 90%, 91%, 92%, 93%, or 94% and even more typically at least 95%, 96%, 97%, 98% or 99% sequence identity to the NTHi polynucleotides recited above.

Included within the scope of the nucleic acid sequences of the invention are nucleic acid sequence fragments that hybridize under stringent conditions to the NTHi nucleotide sequences of SEQ ID NOS: 1-576, SEQ ID NOS: 675-685, SEQ ID NOS: 773-2593, and the nucleotide sequences set out in Table 3B, Table 4B and Table 5 herein, or compliments thereof, which fragment is greater than about 5 nucleotides, preferably 7 nucleotides, more preferably greater than 9 nucleotides and most preferably greater than 17 nucleotides. Fragments of, e.g., 15, 17, or 20 nucleotides or more that are selective for (i.e., specifically hybridize to any one of the polynucleotides of the invention) are contemplated. Probes capable of specifically hybridizing to a polynucleotide can differentiate NTHi polynucleotide sequences of the invention from other polynucleotide sequences in the same family of genes or can differentiate NTHi genes from other bacterial genes, and are preferably based on unique nucleotide sequences.

The term “stringent” is used to refer to conditions that are commonly understood in the art as stringent. Hybridization stringency is principally determined by temperature, ionic strength, and the concentration of denaturing agents such as formamide. Examples of stringent conditions for hybridization and washing are 0.015 M sodium chloride, 0.0015 M sodium citrate at 65-68° C. or 0.015 M sodium chloride, 0.0015M sodium citrate, and 50% formamide at 42° C. See Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^(nd) Ed., Cold Spring Harbor Laboratory, (Cold Spring Harbor, N.Y. 1989). More stringent conditions (such as higher temperature, lower ionic strength, higher formamide, or other denaturing agent) may also be used, however, the rate of hybridization will be affected. In instances wherein hybridization of deoxyoligonucleotides is concerned, additional exemplary stringent hybridization conditions include washing in 6×SSC 0.05% sodium pyrophosphate at 37° C. (for 14-base oligos), 48° C. (for 17-base oligos), 55° C. (for 20-base oligos), and 60° C. (for 23-base oligos).

Other agents may be included in the hybridization and washing buffers for the purpose of reducing non-specific and/or background hybridization. Examples are 0.1% bovine serum albumin, 0.1% polyvinyl-pyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecylsulfate, NaDodSO₄, (SDS), ficoll, Denhardt's solution, sonicated salmon sperm DNA (or other non-complementary DNA), and dextran sulfate, although other suitable agents can also be used. The concentration and types of these additives can be changed without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are usually carried out at pH 6.8-7.4, however, at typical ionic strength conditions, the rate of hybridization is nearly independent of pH. See Anderson et al., Nucleic Acid Hybridisation: A Practical Approach, Ch. 4, IRL Press Limited (Oxford, England). Hybridization conditions can be adjusted by one skilled in the art in order to accommodate these variables and allow DNAs of different sequence relatedness to form hybrids.

The sequences falling within the scope of the present invention are not limited to these specific sequences, but also include allelic and species variations thereof. Allelic and species variations can be routinely determined by comparing the sequence provided in SEQ ID NOS: 1-576, SEQ ID NOS: 675-685, SEQ ID NOS: 773-2593, and nucleotide sequences out in Table 3B, Table 4B and Table 5 herein, preferably the open reading frames therein, a representative fragment thereof, or a nucleotide sequence at least 90% identical, preferably 95% identical, to the open reading frames within SEQ ID NOS: 1-576, SEQ ID NOS: 675-685, SEQ ID NOS: 773-2593, and the nucleotide sequences set out in Table 3B, Table 4B and Table 5 with a sequence from another isolate of the same species. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package, including GAP (Devereux et al., Nucl. Acid. Res., 12: 387, 1984; Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol., 215: 403-410, 1990). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., supra). The well known Smith Waterman algorithm may also be used to determine identity.

Furthermore, to accommodate codon variability, the invention includes nucleic acid molecules coding for the same amino acid sequences as do the specific open reading frames (ORF) disclosed herein. In other words, in the coding region of an ORF, substitution of one codon for another codon that encodes the same amino acid is expressly contemplated.

The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising: the amino acid sequences encoded by the nucleotide sequences included within the polynucleotide sequences set out as SEQ ID NOS: 1-576, SEQ ID NOS: 675-685, SEQ ID NOS: 773-2593, and the nucleotide sequences set out in Table 3B, Table 4B and Table 5, or the corresponding full length or mature protein. The polypeptides of the invention include the amino acid sequences of SEQ ID NO: 616, SEQ ID NO: 618, SEQ ID NO: 620, SEQ ID NO: 622, SEQ ID NO: 624, SEQ ID NO: 626, SEQ ID NO: 628, SEQ ID NO: 628, SEQ ID NO: 630, SEQ ID NO: 632, SEQ ID NO: 634, SEQ ID NO: 636, SEQ ID NO: 638, SEQ ID NO: 640, SEQ ID NO: 642, SEQ ID NO: 644, SEQ ID NO: 646, SEQ ID NO: 648, SEQ ID NO: 650, SEQ ID NO: 652, SEQ ID NO: 654, SEQ ID NO: 656, SEQ ID NO: 658, SEQ ID NO: 660, SEQ ID NO: 662, SEQ ID NO: 664, SEQ ID NO: 666, SEQ ID NO: 668, SEQ ID NO: 670, SEQ ID NO: 672, SEQ ID NO: 674, SEQ ID NO: 687, SEQ ID NO: 689, SEQ ID NO: 691, SEQ ID NO: 693, SEQ ID NO: 695, SEQ ID NO: 697, SEQ ID NO: 699, SEQ ID NO: 701, SEQ ID NO: 703, SEQ ID NO: 705, SEQ ID NO: 707, SEQ ID NO: 709, SEQ ID NO: 711, SEQ ID NO: 713, SEQ ID NO:715, SEQ ID NO: 717, SEQ ID NO: 719, SEQ ID NO: 721, SEQ ID NO:723, SEQ ID NO:725, SEQ ID NO:727, SEQ ID NO:729, SEQ ID NO: 731, SEQ ID NO: 733, SEQ ID NO: 735, SEQ ID NO: 737, SEQ ID NO: 739, SEQ ID NO: 741, SEQ ID NO: 743, SEQ ID NO: 745, SEQ ID NO: 747, SEQ ID NO: 749, SEQ ID NO: 751, SEQ ID NO: 753, SEQ ID NO: 755, SEQ ID NO: 757, SEQ ID NO: 759, SEQ ID NO: 761, 763, SEQ ID NO: 765, SEQ ID NO: 767, SEQ ID NO: 769 or SEQ ID NO: 771, SEQ ID NOS: 2594-4414 which are set out in Table 3B, Table 4B and Table 5 herein.

Polypeptides of the invention also include polypeptides preferably with biological or immunogenic activity that are encoded by: (a) an open reading frame contained within the nucleotide sequences set forth as SEQ ID NOS: 1-576, SEQ ID NOS: 675-685, SEQ ID NOS: 773-2593, and the nucleotide sequences set out in Table 3B, Table 4B and Table 5, or (b) polynucleotides that hybridize to the complement of the polynucleotides of (a) under stringent hybridization conditions.

The invention also provides biologically active or immunologically active variants of the amino acid sequences of the present invention; and “substantial equivalents” thereof (e.g., with at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, 86%, 87%, 88%, 89%, at least about 90%, 91%, 92%, 93%, 94%, typically at least about 95%, 96%, 97%, more typically at least about 98%, or most typically at least about 99% amino acid identity) that retain biological and/or immunogenic activity. Polypeptides encoded by allelic variants may have a similar, increased, or decreased activity compared to polypeptides encoded by the polynucleotides included within the nucleotide sequences presented in SEQ ID NOS: 1-576, SEQ ID NOS: 675-685, SEQ ID NOS: 773-2593, and the nucleotide sequences encoding the amino acid sequences set out as SEQ ID NOS: 2594-4414 and in Table 3B, Table 4B and Table 5 herein, and the polypeptides having an amino acid sequence set out in SEQ ID NOS: 2594-4414, Table 3B, Table 4B and Table 5 herein

NTHi peptides refer to fragments of the NTHi polypeptides encoded by the nucleotide sequences presented in SEQ ID NOS: 1-576, SEQ ID NOS: 675-685, SEQ ID NOS: 773-2593, or the nucleotide sequences set out in Table 3B, Table 4B and Table 5 herein, and the polypeptides having the amino acid sequences set out in SEQ ID NOS: 2594-4414 Table 3B, Table 4B and Table 5 herein. The preferred NTHi peptides are biologically and/or immunologically active.

The present invention further provides isolated NTHi polypeptides or NTHi peptides encoded by the NTHi nucleic acid fragments of the present invention or by degenerate variants of the nucleic acid fragments of the present invention. The term “degenerate variant” refers to nucleotide fragments which differ from a nucleic acid fragment of the present invention (e.g., an ORF) by nucleotide sequence but, due to the degeneracy of the genetic code, encode an identical NTHi polypeptide sequence. Preferred nucleic acid fragments of the present invention are the ORFs that encode proteins.

The invention also provides for NTHi polypeptides with one or more conservative amino acid substitutions that do not affect the biological and/or immunogenic activity of the polypeptide. Alternatively, the NTHi polypeptides of the invention are contemplated to have conservative amino acids substitutions which may or may not alter biological activity. The term “conservative amino acid substitution” refers to a substitution of a native amino acid residue with a normative residue, including naturally occurring and nonnaturally occurring amino acids, such that there is little or no effect on the polarity or charge of the amino acid residue at that position. For example, a conservative substitution results from the replacement of a non-polar residue in a polypeptide with any other non-polar residue. Further, any native residue in the polypeptide may also be substituted with alanine, according to the methods of “alanine scanning mutagenesis”. Naturally occurring amino acids are characterized based on their side chains as follows: basic: arginine, lysine, histidine; acidic: glutamic acid, aspartic acid; uncharged polar: glutamine, asparagine, serine, threonine, tyrosine; and non-polar: phenylalanine, tryptophan, cysteine, glycine, alanine, valine, proline, methionine, leucine, norleucine, isoleucine General rules for amino acid substitutions are set forth in Table 1 below.

TABLE 1 Amino Acid Substitutions Original Residues Exemplary Substitutions Preferred Substitutions Ala Val, Leu, Ile Val Arg Lys, Gln, Asn Lys Asn Gln Gln Asp Glu Glu Cys Ser, Ala Ser Gln Asn Asn Glu Asp Asn Gly Pro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Phe, Leu Leu Norleucine, Ile, Val, Met, Leu Lys Arg, 1,4 Diaminobutyric Arg Met Leu, Phe, Ile Leu Phe Leu, Val, Ile, Ala, Tyr Arg Pro Ala Gly Ser Thr, Ala, Cys Thr Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Met, Leu, Phe, Ala, Leu

Antisense polynucleotides complementary to the polynucleotides encoding the NTHi polypeptides are also provided.

The invention contemplates that polynucleotides of the invention may be inserted in a vector for amplification or expression. For expression, the polynucleotides are operatively linked to appropriate expression control sequence such as a promoter and polyadenylation signal sequences. Further provided are cells comprising polynucleotides of the invention. Exemplary prokaryotic hosts include bacteria such as E. coli, Bacillus, Streptomyces, Pseudomonas, Salmonella and Serratia.

The term “isolated” refers to a substance removed from, and essentially free of, the other components of the environment in which it naturally exists. For example, a polypeptide is separated from other cellular proteins or a DNA is separated from other DNA flanking it in a genome in which it naturally occurs.

Antibodies and Methods for Eliciting an Immune Response

The invention provides antibodies which bind to antigenic epitopes unique to (i.e., are specific for) NTHi polypeptides. Also provided are antibodies which bind to antigenic epitopes common among multiple H. influenzae subtypes but unique with respect to any other antigenic epitopes. The antibodies may be polyclonal antibodies, monoclonal antibodies, antibody fragments which retain their ability to bind their unique epitope (e.g., Fv, Fab and F(ab)2 fragments), single chain antibodies and human or humanized antibodies. Antibodies may be generated by techniques standard in the art.

It is known in the art that antigens that mimic the capsular polysaccharide of H. influenzae will generate antibodies that exhibit the ability to kill bacteria in in vitro assays. These antibodies are also known to protect against challenge with H. influenzae in animal model systems. These studies indicate antibody to the capsular polysacchamides are likely to elicit a protective immune response in humans. The present invention provides for antibodies specific for the NTHi polypeptides of the present invention and fragments thereof, which exhibit the ability to kill both H. influenzae bacteria and to protect humans from NTHi infection. The present invention also provides for antibodies specific for the NTHi polypeptides of the invention which reduce the virulence, inhibit adherence, inhibit cell division, and/or inhibit penetration into the epithelium of H. influenzae bacteria or enhance phagocytosis of the H. influenzae bacteria.

In vitro complement mediated bactericidal assay systems (Musher et al., Infect. Immun. 39: 297-304, 1983; Anderson et al., J. Clin. Invest. 51: 31-38, 1972) may be used to measure the bactericidal activity of anti-NTHi antibodies. Further data on the ability of NTHi polypeptides and NTHi peptides to elicit a protective antibody response may be generated by using animal models of infection such as the chinchilla model system described herein.

It is also possible to confer short-term protection to a host by passive immunotherapy via the administration of pre-formed antibody against an epitope of NTHi, such as antibodies against NTHi OMP, LOS or noncapsular proteins. Thus, the contemplated vaccine formulations can be used to produce antibodies for use in passive immunotherapy. Human immunoglobulin is preferred in human medicine because a heterologous immunoglobulin may provoke an immune response to its foreign immunogenic components. Such passive immunization could be used on an emergency basis for immediate protection of unimmunized individuals exposed to special risks. Alternatively, these antibodies can be used in the production of anti-idiotypic antibody, which in turn can be used as an antigen to stimulate an immune response against NTHi epitopes.

The invention contemplates methods of eliciting an immune response to NTHi in an individual. These methods elicit immune responses which include one or more of killing NTHi bacteria, blocking NTHi attachment to cells and/or slowing NTHi replication. An “immunogenic dose” of a composition of the invention is one that generates, after administration, a detectable humoral and/or cellular immune response in comparison to the immune response detectable before administration or in comparison to a standard immune response before administration. The invention contemplates that the immune response resulting from the methods may be protective and/or therapeutic. In one embodiment, the methods comprise a step of administering an immunogenic dose of a composition comprising a NTHi protein or NTHi peptide of the invention. A NTHi protein or an antigenic peptide thereof may be fused with co-protein which may not by itself produce antibodies, but is capable of stabilizing the first protein and producing a fused protein which will have immunogenic and protective properties. Thus fused recombinant protein, preferably further comprises an antigenic co-protein, such as Glutathione-S-transferase (GST) or beta-galactosidase, relatively large co-proteins which solubilize the protein and facilitate production and purification thereof. Moreover, the co-protein may act as an adjuvant in the sense of providing a generalized stimulation of the immune system. The co-protein may be attached to either the amino or carboxy terminus of the first protein. Provided by this invention are compositions, particularly vaccine compositions, and methods comprising the NTHi polypeptides encoded by the polynucleotide of the invention or antigenic peptides thereof.

The invention also contemplates methods of eliciting an immune response to multiple H. influenzae subtypes in an individual. These methods elicit immune responses which include one or more of killing the H. influenzae bacteria, blocking H. influenzae attachment to cells and/or slowing H. influenzae replication. These methods comprise a step of administering an immunogenic dose of a composition comprising a NTHi protein or NTHi peptide of the invention which comprise an antigenic epitope that is common among multiple H. influenzae subtypes but unique with respect to any other antigenic epitopes.

In another embodiment, the methods comprise administering an immunogenic dose of a composition comprising a cell expressing a NTHi protein or NTHi peptide of the invention. In yet another embodiment, the methods comprise administering an immunogenic dose of a composition comprising a polynucleotide encoding a NTHi protein or NTHi peptide of the invention. The polynucleotide may be a naked polynucleotide not associated with any other nucleic acid or may be in a vector such as a plasmid or viral vector (e.g., adeno-associated virus vector or adenovirus vector). The compositions of the invention may comprise one or more NTHi proteins or NTHi peptides alone or in combination with other epitopes that elicit an immune response to NTHi bacteria. The compositions of the invention may also comprise one or more NTHi proteins or peptides in combination with epitopes that elicit an immune response to one or more H. influenzae subtypes. Administration of the compositions may be by routes standard in the art, for example, parenteral, intravenous, oral, buccal, nasal, pulmonary, rectal, or vaginal. The methods may be used in combination in a single individual. The methods may be used prior or subsequent to NTHi infection of an individual.

Genes that are up-regulated in NTHi infection of the middle ear and/or the nasopharynx and genes that are associated with NTHi virulence are described herein. The polypeptides and peptides thereof which are encoded by these NTHi genes are contemplated to be useful for eliciting an immune response for treating or preventing disorders associated with NTHi infection, such as OM. Some of the polypeptides encoded by these genes include: histidine biosynthesis protein, lipoprotein B, peptide ABC transporter, periplasmic SapA precursor, outer membrane lipoproteins carrier protein precursor, ribose transport system permease protein, phosphoribosylaminoimidazole carboxylase catalytic subunit, PurE, Phosphoribosylaminoimidazole carboxylase catalytic subunit, ornithine carbamolytransferase, mannonate dehydratase, disulfide oxidoreductase, urease accessory protein, phospshocholine cytidylytransferase, putative pyridoxine biosynthesis protein, singlet oxygen resistance protein, intracellular septation protein, DNA repair protein, MukF protein, glycerol-3-phosphate regulon repressor, integration host factor beta subunit, arginine repressor, cold shock like protein, stress response protein, LicA, MukF, RadA and those hypothetical proteins encoded by HI0094, HI1163, HI0665, HI1292, HI1064 HI186, HI0352 genes. NTHi OMPs, LOS and noncapsular proteins are also contemplated to elicit an immune response for prevention and treatment of disorders associated with NTHi infection.

The invention includes methods of blocking binding of NTHi bacteria to host cells in an individual. The methods comprise administering antibodies or polypeptides of the invention that block binding of NTHi cellular attachment. Alternatively, administration of one or more small molecules that block binding of NTHi cell attachment is contemplated. In vitro assays may be used to demonstrate the ability of an antibody, polypeptide or small molecule of the invention to block NTHi cell attachment.

Pharmaceutical compositions comprising antibodies of the invention, polypeptides of the invention and/or small molecules of the invention that block NTHi cellular attachment are provided. The pharmaceutical compositions may consist of one of the foregoing active ingredients alone, may comprise combinations of the foregoing active ingredients or may comprise additional active ingredients used to treat bacterial infections. The pharmaceutical compositions may comprise one or more additional ingredients such as pharmaceutically effective carriers. Dosage and frequency of the administration of the pharmaceutical compositions are determined by standard techniques and depend, for example, on the weight and age of the individual, the route of administration, and the severity of symptoms. Administration of the pharmaceutical compositions may be by routes standard in the art, for example, parenteral, intravenous, oral, buccal, nasal, pulmonary, rectal, or vaginal.

Also provided by the invention are methods for detecting NTHi infection in an individual. In one embodiment, the methods comprise detecting NTHi polynucleotides of the invention in a sample using primers or probes that specifically bind to the polynucleotides. Detection of the polynucleotide may be accomplished by numerous techniques routine in the art involving, for example, hybridization and PCR.

The antibodies of the present invention may also be used to provide reagents for use in diagnostic assays for the detection of NTHi antigens (NTHi polypeptides and peptides thereof) in various body fluids of individuals suspected of H. influenzae infection. In another embodiment, the NTHi proteins and peptides of the present invention may be used as antigens in immunoassays for the detection of NTHi in various patient tissues and body fluids including, but not limited to: blood, serum, ear fluid, spinal fluid, sputum, urine, lymphatic fluid and cerebrospinal fluid. The antigens of the present invention may be used in any immunoassay system known in the art including, but not limited to: radioimmunoassays, ELISA assays, sandwich assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, fluorescent immunoassays, protein A immunoassays and immunoelectrophoresis assays.

Vaccines and Chemotherapeutic Targets

An aspect of the invention relates to a method for inducing an immunological response in an individual, particularly a mammal which comprises inoculating the individual with a NTHi antigen protein or an antigenic peptide thereof.

The present invention also provides for vaccine formulations which comprise an immunogenic recombinant NTHi protein or NTHi peptide of the invention together with a suitable carrier. The NTHi polypeptides and peptides thereof contemplated as vaccine candidates and/or targets of chemotherapy include, but are not limited to, histidine biosynthesis protein, lipoprotein B, peptide ABC transporter, periplasmic SapA precursor, outer membrane lipoproteins carrier protein precursor, ribose transport system permease protein, phosphoribosylaminoimidazole carboxylase catalytic subunit, PurE, 3,4-dihydroxt-2-butone 4-phosphate synthase, ornithine carbamolytransferase, mannonate dehydratase, disulfide oxidoreductase, urease accessory protein, phospshocholine cytidylytransferase, putative pyridoxine biosynthesis protein, singlet oxygen resistance protein, intracellular septation protein, DNA repair protein, MUKF protein, glycerol-3-phosphate regulon repressor, integration host factor beta subunit, arginine repressor, cold shock like protein, stress response protein, LicA, RadA and those hypothetical proteins encoded by HI0094, HI1163, HI0665, HI1292, HI1064 HI1386, HI0352 genes, NTHi OMPs, NTHi LOS and NTHi noncapsular proteins and polypeptides encoded by the novel NTHi polynucleotide sequences present in the nucleotide sequences set out as SEQ ID NOS: 1-576, SEQ ID NOS: 675-685, SEQ ID NOS: 773-2593, and the nucleotide sequences set out in Table 3B, Table 4B and Table 5 herein, and the polypeptides having the amino acid sequences set out in SEQ ID NOS: 2594-4414, Table 3B, Table 4B and Table 5 herein.

Since the protein may be broken down in the stomach, it is preferably administered parenterally, including, for example, administration that is subcutaneous, intramuscular, intravenous, or intradermal. Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the bodily fluid, preferably the blood, of the individual; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use. The vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.

A. Peptide Vaccines

Peptide therapeutic agents, such as peptide vaccines, are well known in the art and are of increasing use in the pharmaceutical arts. Consistent drawbacks to the parenteral administration of such peptide compounds have been the rapidity of breakdown or denaturation. Infusion pumps, as well as wax or oil implants, have been employed for chronic administration of therapeutic agents in an effort to both prolong the presence of peptide-like therapeutic agents and preserve the integrity of such agents. Furthermore, the peptide-like agent should (with particular reference to each epitope of the peptide-like agent) ideally maintain native state configuration for an extended period of time and additionally be presented in a fashion suitable for triggering an immunogenic response in the challenged animal or immunized human.

The NTHi antigenic peptides of the invention can be prepared in a number of conventional ways. The short peptides sequences can be prepared by chemical synthesis using standard means. Particularly convenient are solid phase techniques (see, e.g., Erikson et al., The Proteins (1976) v. 2, Academic Press, New York, p. 255). Automated solid phase synthesizers are commercially available. In addition, modifications in the sequence are easily made by substitution, addition or omission of appropriate residues. For example, a cysteine residue may be added at the carboxy terminus to provide a sulfhydryl group for convenient linkage to a carrier protein, or spacer elements, such as an additional glycine residue, may be incorporated into the sequence between the linking amino acid at the C-terminus and the remainder of the peptide. The short NTHi peptides can also be produced by recombinant techniques. The coding sequence for peptides of this length can easily be synthesized by chemical techniques, e.g., the phosphotriester method described in Matteucci et al., J Am Chem. Soc., 103: 3185 (1981).

Some of the NTHi peptide sequences contemplated herein may be considered too small to be immunogenic, they may be linked to carrier substances in order to confer this property upon them. Any method of creating such linkages known in the art may be used. Linkages can be formed with heterobifunctional agents that generate a disulfide link at one functional group end and a peptide link at the other, such as a disulfide amide forming agent, e.g., N-succidimidyl-3-(2-pyridyldithio) proprionate (SPDP) (See, e.g., Jansen et al., Immun. Rev. 62:185, 1982) and bifunctional coupling agents that form a thioether rather than a disulfide linkage such as reactive esters of 6-maleimidocaproic acid, 2-bromoacetic acid, 2-iodoacetic acid, 4-(N-maleimido-methyl)cyclohexane-1-carboxylic acid and the like, and coupling agent which activate carboxyl groups by combining them with succinimide or 1-hydroxy-2-nitro-4-sulfonic acid, for sodium salt such as succinimmidyl 4-(N-maleimido-methyl)cyclohexane-1-carobxylate (SMCC).

B. Vaccine Compositions and Administration

A priming dose of the immunogen that is followed by one or more booster exposures to the immunogen may be necessary to be an effective vaccine (Kramp et al., Infect. Immun., 25: 771-773, 1979; Davis et al., Immunology Letters, 14: 341-8 1986 1987). Examples of proteins or polypeptides that could beneficially enhance the immune response if co-administered include cytokines (e.g., IL-2, IL-12, GM-CSF), cytokine-inducing molecules (e.g. Leaf) or costimulatory molecules. Helper (HTL) epitopes could be joined to intracellular targeting signals and expressed separately from the CTL epitopes. This would allow direction of the HTL epitopes to a cell compartment different than the CTL epitopes. If required, this could facilitate more efficient entry of HTL epitopes into the MHC class II pathway, thereby improving CTL induction. In contrast to CTL induction, specifically decreasing the immune response by co-expression of immunosuppressive molecules (e.g. TGF-β) may be beneficial in certain diseases.

Ideally, an immunogen will exhibit two properties; the capacity to stimulate the formation of the corresponding antibodies and the propensity to react specifically with these antibodies. Immunogens bear one or more epitopes which are the smallest part of an immunogen recognizable by the combing site of an antibody. In particular instances, immunogen, fractions of immunogens or conditions under which the immunogen is presented are inadequate to precipitate the desired immunological response resulting in insufficient immunity. This is often the case with peptides or other small molecules used as immunogens. Other substances such as immunomodulators (e.g., cytokines such as the interleukins) may be combined in vaccines as well.

The vaccine art recognizes the use of certain substances called adjuvants to potentate an immune response when used in conjunction with an immunogen. Adjuvants are further used to elicit an immune response that is faster or greater than would be elicited without the use of the adjuvant. In addition, adjuvants may be used to create an immunological response using less immunogen than would be needed without the inclusion of adjuvant, to increase production of certain antibody subclasses that afford immunological protection or to enhance components of the immune response (e.g., humoral, cellular). Known adjuvants include emulsions such as Freund's Adjuvants and other oil emulsions, Bordetella pertussis, MF59, purified saponin from Quillaja saponaria (QS21), aluminum salts such as hydroxide, phosphate and alum, calcium phosphate, (and other metal salts), gels such as aluminum hydroxide salts, mycobacterial products including muramyl dipeptides, solid materials, particles such as liposomes and virosomes. Examples of natural and bacterial products known to be used as adjuvants include monophosphoryl lipid A (MPL), RC-529 (synthetic MPL-like acylated monosaccharide), OM-174 which is a lipid A derivative from E. coli, holotoxins such as cholera toxin (CT) or one of its derivatives, pertussis toxin (PT) and heat-labile toxin (LT) of E. coli or one of its derivatives, and CpG oligonucleotides. Adjuvant activity can be affected by a number of factors, such as carrier effect, depot formation, altered lymphocyte recirculation, stimulation of T-lymphocytes, direct stimulation of B-lymphocytes and stimulation of macrophages.

Vaccines are typically prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified. The active immunogenic ingredient is often mixed with excipients, which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, e.g., water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants, which enhance the effectiveness of the vaccine. The vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include, for example, polyalkalene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1-2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%-95% of active ingredient, preferably 25-70%.

Vaccines may also be administered through transdermal routes utilizing jet injectors, microneedles, electroporation, sonoporation, microencapsulation, polymers or liposomes, transmucosal routes and intranasal routes using nebulizers, aerosols and nasal sprays. Microencapsulation using natural or synthetic polymers such as starch, alginate and chitosan, D-poly L-lactate (PLA), D-poly DL-lactic-coglycolic microspheres, polycaprolactones, polyorthoesters, polyanhydrides and polyphosphazenes polyphosphatazanes are useful for both transdermal and transmucosal administration. Polymeric complexes comprising synthetic poly-ornithate, poly-lysine and poly-arginine or amphipathic peptides are useful for transdermal delivery systems. In addition, due to their amphipathic nature, liposomes are contemplated for transdermal, transmucosal and intranasal vaccine delivery systems. Common lipids used for vaccine delivery include N-(1)2,3-(dioleyl-dihydroxypropyl)-N,N,N,-trimethylammonium-methyl sulfate (DOTAP), dioleyloxy-propyl-trimethylammonium chloride DOTMA, dimystyloxypropyl-3-dimethyl-hydroxyethyl ammonium (DMRIE), dimethyldioctadecyl ammonium bromide (DDAB) and 9N(N′,N-dimethylaminoethane) carbamoyl) cholesterol (DC-Chol). The combination of helper lipids and liposomes will enhance up-take of the liposomes through the skin. These helper lipids include, dioleoyl phosphatidylethanolamine (DOPE), dilauroylphosphatidylethanolamine (DLPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE). In addition, triterpenoid glycosides or saponins derived from the Chilean soap tree bark (Quillaja saponaria) and chitosan (deacetylated chitan) have been contemplated as useful adjuvants for intranasal and transmucosal vaccine delivery.

The proteins may be formulated into the vaccine as neutral or salt forms. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the peptide) and which are formed with inorganic acids such as, e.g., hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, and procaine.

The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic. The quantity to be administered depends on the subject to be treated, capacity of the subject's immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosage ranges are of the order of several hundred micrograms active ingredient per individual. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed in one or three month intervals by a subsequent injection or other administration.

Upon immunization with a vaccine composition as described herein, the immune system of the host responds to the vaccine by producing large amounts of CTLs specific for the desired antigen, and the host becomes at least partially immune to later infection, or resistant to developing chronic infection. Vaccine compositions containing the NTHi polypeptide or NTHi peptides of the invention are administered to a patient susceptible to or otherwise at risk of bacterial infection to elicit an immune response against the antigen and thus enhance the patient's own immune response capabilities. In this use, the precise amounts again depend on the patient's state of health and weight, the mode of administration, the nature of the formulation, etc., but generally range from about 1.0 μg to about 5000 per 70 kilogram patient, more commonly from about 10 to about 500 mg per 70 kg of body weight. For therapeutic or immunization purposes, the NTHi polypeptide or NTHi peptides of the invention can also be expressed by attenuated viral hosts, such as vaccinia or fowlpox. This approach involves the use of vaccinia virus as a vector to express nucleotide sequences that encode the peptides of the invention. Upon introduction into an acutely or chronically infected host or into a noninfected host, the recombinant vaccinia virus expresses the immunogenic peptide, and thereby elicits a host CTL response.

Humoral immune response may be measured by many well known methods, such as Single Radial Immunodiffussion Assay (SRID), Enzyme Immunoassay (EIA) and Hemagglutination Inhibition Assay (HAI). In particular, SRID utilizes a layer of a gel, such as agarose, containing the immunogen being tested. A well is cut in the gel and the serum being tested is placed in the well. Diffusion of the antibody out into the gel leads to the formation of a precipitation ring whose area is proportional to the concentration of the antibody in the serum being tested. EIA, also known as ELISA (Enzyme Linked Immunoassay), is used to determine total antibodies in the sample. The immunogen is adsorbed to the surface of a microtiter plate. The test serum is exposed to the plate followed by an enzyme linked immunoglobulin, such as IgG. The enzyme activity adherent to the plate is quantified by any convenient means such as spectrophotometry and is proportional to the concentration of antibody directed against the immunogen present in the test sample. HAI utilizes the capability of an immunogen such as viral proteins to agglutinate chicken red blood cells (or the like). The assay detects neutralizing antibodies, i.e., those antibodies able to inhibit hemagglutination. Dilution of the test serum are incubated with a standard concentration of immunogen, followed by the addition of the red blood cells. The presence of neutralizing antibodies will inhibit the agglutination of the red blood cells by the immunogen. Tests to measure cellular immune response include determination of delayed-type hypersensitivity or measuring the proliferative response of lymphocytes to target immunogen.

Nontypeable Haemophilus influenzae (NTHi)

H. influenzae is a small, nonmotile gram negative bacterium. Unlike other H. influenzae strains, the nontypeable H. influenzae (NTHi) strains lack a polysaccharide capsule and are sometimes denoted as “nonencapsulated.” NTHi strains are genetically distinct from encapsulated strains and are more heterogenous than the type b H. influenzae isolates. NTHi presents a complex array of antigens to the human host. Possible antigens that may elicit protection include OMPs, lipopolysaccharides, lipoproteins, adhesion proteins and noncapsular proteins.

Humans are the only host for H. influenze. NTHi strains commonly reside in the upper respiratory tract including the nasopharynx and the posterior oropharynx, the lower respiratory tract and the female genital tract. NTHi causes a broad spectrum of diseases in humans, including but not limited to, otitis media, pneumonia, sinusitis, septicemia, endocarditis, epiglottitis, septic arthritis, meningitis, postpartum and neonatal infections, postpartum and neonatal sepsis, acute and chromic salpingitis, epiglottis, pericarditis, cellulitis, osteomyelitis, endocarditis, cholecystitis, intraabdominal infections, urinary tract infection, mastoiditis, aortic graft infection, conjunctitivitis, Brazilian purpuric fever, occult bacteremia and exacerbation of underlying lung diseases such as chronic bronchitis, bronchietasis and cystic fibrosis.

Epidemiologic studies of NTHi have indicated that the strains are heterogeneous with respect to outer membrane protein profiles (Barenkamp et al., Infect. Immun., 36: 535-40, 1982), enzyme allotypes (Musser et al., Infect. Immun., 52: 183-191, 1986), and other commonly used epidemiologic tools. There have been several attempts to subtype NTHi, but none of the methodologies have been totally satisfactory. The outer-membrane protein composition of NTHi consists of approximately 20 proteins. All NTHi strains contains two common OMP's with molecular weights of 30,000 and 16,600 daltons. NTHi strains may be subtyped based on two OMP's within the 32,000-42,000 dalton range. The NTHi liposaccharide profile is fundamentally different than the enteric gram negative bacteria and separates into 1-4 distinct bands ranging from less than 20,000 daltons.

A prototype NTHi isolate is the low passage isolate 86-028NP which was recovered from a child with chronic otitis media. This strain has been well characterized in vitro (Bakaletz et al., Infect. Immun., 53: 331-5, 1988; Holmes et al., Microb. Pathog., 23: 157-66, 1997) as well as in the chinchilla OM model (described herein) (Bakaletz et al., Vaccine, 15: 955-61, 1997; Suzuki et al., Infect. Immun., 62: 1710-8, 1994; DeMaria et al., Infect. Immun., 64: 5187-92, 1996). The 86-028NP strain was used, as described herein, to identify genes that are up-regulated in expression in the chinchilla model of otitis media and genes that are necessary for NTHi survival in the chinchilla middle ear.

DFI Strategy

A differential fluorescence induction (DFI) strategy was used herein to identify NTHi genes induced during OM in a chinchilla animal model. Several methods have been developed to identify bacterial genes that contribute to the virulence of an organism during infection. Such methods include in vivo expression technology (IVET) in which bacterial promoters regulate the expression of gene(s) required for synthesis of essential nutrients required for survival in the host; signature-tagged mutagenesis (STM) enabling tag-specific identification of genes that alter the virulence properties of a microorganism when mutated; DNA microarray technology to globally screen for transcriptionally active genes, and DFI which uses FACS analysis to select for transcriptionally active promoters (Chiang et al., Annu. Rev. Microbiol., 53: 129-154, 1999). DFI is a high-throughput method that allows for the identification of differentially regulated genes regardless of the basal level of expression and does not exclude those that are essential for growth in vitro.

DFI has been successfully utilized in many microorganisms. For example, a GFP reporter system and flow cytometry was used to study mycobacterial gene expression upon interaction with macrophages (Dhandayuthapani et al., Mol. Microbiol., 17: 901-912, 1995). A promoter trap system was used to identify genes whose transcription was increased when Salmonellae were subjected to environments simulating in vivo growth and when internalized by cultured macrophage-like cells (Valdivia and Falkow, Mol. Microbiol., 22: 367-378, 1996; Valdivia and Falkow, Science, 277: 2007-2011, 1997; Valdivia and Falkow, Curr. Opin. Microbiol., 1: 359-363, 1998). In addition, DFI has been used to identify promoters expressed in S. pneumoniae and S. aureus when grown under varied in vitro conditions simulating infection (Marra et al., Infect. Immun., 148: 1483-1491, 2002; Schneider et al., Proc. Natl. Acad. Sci. U.S.A., 97: 1671-1676, 2000). In addition, DFI has been utilized to study gene regulation in Bacillus cereus in response to environmental stimuli (Dunn and Handelsman, Gene, 226: 297-305, 1999), in S. pneumoniae in response to a competence stimulatory peptide (Bartilson et al., Mol. Microbiol., 39: 126-135, 2001), and upon interaction with and invasion of host cells in Bartonella henselae Lee and Falkow, Infect. Immun., 66: 3964-3967, 1998), Listeria monocytogenes Wilson et al., Infect. Immun., 69: 5016-5024, 2001), Brucella abortus (Eskra et al., Infect. Immun., 69: 7736-7742, 2001), and Escherichia coli (Badger et al., Mol. Microbiol., 36: 174-182, 2000).

Whereas DFI has been successfully used to identify promoters active in cell culture models of infection or in vitro conditions designed to simulate an in vivo environment, few have applied DFI to identify promoters regulated in a specific biological niche within the whole animal. This is likely due to the numerous challenges associated with sorting from an in vivo environment. The host inflammatory response, dissemination and/or clearance of bacterial cells from the site of infection, as well as adherence of bacteria to epithelial cells, possibly via biofilm formation, can make bacteria inaccessible for retrieval from the living animal. These factors, among others, contribute to the complexity of the microenvironment and the heterogeneity of gene expression as the bacteria sense and respond to these changes. Recently, DFI has been used to identify promoters expressed in S. pneumoniae when the bacteria were screened in a mouse model of respiratory tract infection and a gerbil infection model of OM (Marra et al., Infect. Immun. 70: 1422-33, 2002; Marra et al., Microbiol., 148: 1483-91, 2002).

Animal Model

The chinchilla model is a widely accepted experimental model for OM. In particular, a chinchilla model of NTHi-induced OM has been well characterized (Bakaletz et al., J. Infect. Dis., 168: 865-872, 1993; Bakaletz and Holmes, Clin. Diagn. Lab. Immunol., 4: 223-225, 1997; Suzuki and Bakaletz, Infect. Immun., 62: 1710-1718, 1994), and has been used to determine the protective efficacy of several NTHi outer membrane proteins, combinations of outer membrane proteins, chimeric synthetic peptide vaccine components, and adjuvant formulations as vaccinogens against OM (Bakaletz et al., Vaccine, 15: 955-961, 1997; Bakaletz et al., Infect. Immun., 67: 2746-2762, 1999; Kennedy et al., Infect. Immun., 68: 2756-2765, 2000).

In particular, there is an unique in vivo model wherein adenovirus predisposes chinchillas to H. influenzae-induced otitis media, which allowed for the establishment of relevant cell, tissue and organ culture systems for the biological assessment of NTHi (Bakaletz et al., J. Infect. Dis., 168: 865-72, 1993; Suzuki et al., Infect. Immunity 62: 1710-8, 1994). Adenovirus infection alone has been used to assess for the transudation of induced serum antibodies into the tympanum (Bakaletz et al., Clin. Diagnostic Lab Immunol., 4(2): 223-5, 1997) and has been used as a co-pathogen with NTHi, to determine the protective efficacy of several active and passive immunization regimens targeting various NTHi outer membrane proteins, combinations of OMPs, chimeric synthetic peptide vaccine components, and adjuvant formulations as vaccinogens against otitis media (Bakaletz et al., Infect Immunity, 67(6): 2746-62, 1999; Kennedy et al., Infect Immun., 68(5): 2756-65, 2000; Novotny et al., Infect Immunity 68(4): 2119-28, 2000; Poolman et al., Vaccine 19 (Suppl. 1): S108-15, 2000).

Genes Unregulated In Vivo in Response to Nthi Infection of the Middle Ear

In order to identify differentially regulated promoters in response to NTHi infection of the middle ear, a promoter trap library was constructed and sorting parameters defined. A portion of the promoter trap library was inoculated directly into the chinchilla middle ear and OM development was monitored by video otoscopy and tympanometry at 24 and 48 hours. In addition, the middle ear fluids were recovered 24 and 48 hours after infection. Two-color FACS analysis was used to isolated bacteria that were expressing GFP from other cells and debris associated with the effusion. Following isolation, DNA sequence of the Haemophilus inserts 5′ of the gfpmut3 gene were determined and analyzed. In this manner, we identified genes that are up-regulated as NTHi sense and respond to the environment of the chinchilla middle ear during AOM. The following genes were identified and due to their up-regulation during NTHi infection, they may play a role in NTHi infection and virulence.

As described below in Example 7, following the DFI procedure described above and subsequent FACS analysis of gfp-expressing clones, 52 candidate clones containing potential in vivo-regulated promoters were isolated. The genes these clones control were categorized based upon general description and function within the cell and include general metabolic processes, environmental informational processing and membrane transport, membrane proteins and hypothetical proteins. Eight of these 52 clones contain sequences that are unique to NTHi strain 86-028NP. Importantly, 3 clones were isolated from independent screens in more than one animal thereby verifying the method of isolation.

In order to independently confirm the FACS data, we determined the relative expression of candidate genes by quantitative RT-PCR. The parent strain 86-028NP, was used for these studies. Thus, wild-type gene expression without the influence of plasmid copy number on gene regulation was analyzed, allowing for the indication of false-positive clone identification by FACS. Of the 44 candidate clones containing sequence similar to that identified in H. influenzae strain Rd, quantitative comparison of gene expression in vitro and in vivo confirmed up-regulated gene expression for twenty-six genes (60%) when NTHi respond to environmental cues present in the chinchilla middle ear. This analysis identified in vivo-regulated promoters which drive expression of genes involved in membrane transport, environmental informational processing, cellular metabolism, gene regulation, as well as hypothetical proteins with unknown function. (See Table 4 in Example 6).

Quantitative RT-PCR demonstrated a two-fold increase in lolA expression, enabling lipoprotein transport from the inner membrane to the outer membrane. Bacteria grow rapidly in the middle ear environment reaching 5.0×10⁸ CFU NTHi ml middle ear fluid within 48 hours. The bacteria sense and respond to the environment, acquiring or synthesizing the necessary nutrients for growth and survival. The gene encoding the membrane component in ribose sugar transport, rbsC (SEQ ID NO: 619), showed a 5-fold increase in expression in vivo compared to cells growing in vitro. In addition, many genes involved in metabolic processes show a dramatic increase in gene expression in vivo compared to cells growing in vitro. These include a riboflavin synthesis gene, ribB (SEQ ID NO: 623), a purine nucleotide biosynthetic gene purE (SEQ ID NO: 621), ornithine carbamoyltransferase, arcB (SEQ ID NO: 625), involved in arginine degradation via the urea cycle and uxuA (SEQ ID NO: 627), encoding mannonate hydrolase, required for the uptake of D-glucuronate and transformation into glyceraldehyde 3-phosphate. In addition, but to a lesser degree, genes for histidine biosynthesis (hisB; SEQ ID NO: 615), DNA repair (radC; SEQ ID NO: 639) and a putative intracellular septation transmembrane protein (ispZ; SEQ ID NO: 637) were up-regulated.

Disulfide bond formation is important for folding and assembly of many secreted proteins in bacteria. In prokaryotes, DsbA and DsbB make up the oxidative pathway responsible for the formation of disulfides. DsbB reoxidizes DsbA, which donates disulfide bonds directly to unfolded polypeptides, and DsbB has been demonstrated to generate disulfides de novo from oxidized quinones (Collet and Bardwell, Mol. Microbiol., 44: 1-8, 2002). In H. influenzae strain Rd, DsbA is required for competence for transformation (Tomb, Proc. Natl. Acad. Sci. U.S.A., 89: 10252-10256, 1992). Herein, an approximate 3-fold increase in dsbB gene (SEQ ID NO: 629) transcription was demonstrated, illuminating an important role for disulfide interchange for NTHi growing in the middle ear environment.

Bacteria colonization of the middle ear, a normally sterile environment, results in a host inflammatory response and subsequent neutrophil infiltration. Bacteria have evolved numerous strategies to combat this host response. NTHi increase gene expression (4-fold) of ureH (SEQ ID NO:631), a homologue of a gene required for expression of active urease in Helicobacter, shown to be involved in acid tolerance (Young et al., J. Bacterol., 178: 6487-6495, 1996). Recently, it has been reported that urease activity may play a role in chronic Actinobacillus pleuropneumoniae infection by counteracting the decrease in pH occurring upon infection (Baltes et al., Infect. Immun., 69: 472-478, 2000; Baltes et al., Infect. Immun., 69: 472-478, 2001; Bosse and MacInnes, Can. J. Vet. Res., 64: 145-150). A biotype analysis on NTHi isolates from middle ear effusions demonstrated that 87% are urease positive (DeMaria et al., J. Clin. Microbiol., 20: 1102-1104, 1984). However, the role of urease in NTHi virulence is unknown. Similarly, an increase in expression of a gene whose product demonstrates 88% sequence identity to a pyridoxine biosynthesis protein in S. pneumoniae and 60% homology to a putative singlet oxygen resistance protein that may function as an antioxidant. Phosphorylcholine (ChoP) has been implicated in the pathogenesis of NTHi (Weiser et al., Infect. Immun., 65: 943-950, 1997). NTHi modulates ChoP expression by phase variation, decorating the LOS on the cell surface. ChoP may contribute to NTHi persistence in the respiratory tract via decreased susceptibility to antimicrobial peptides (Lysecko et al., Infect. Immun., 68: 1664-1671, 2000) and alter the sensitivity to serum killing mediated by C-reactive protein (CRP) (Weiser et al., J. Exp. Med., 187: 631-640, 1998). The microenvironment of the nasopharynx and middle ear cavity may select for the ChoP phenotype, as ChoP strains show greater colonization of the chinchilla nasopharynx (Tong et al., Infect. Immun., 68: 4593-4597, 2000). Expression of the licC gene (SEQ ID NO: 633) was also increased. The licC gene encodes a phosphorylcholine cytidylyltransferase that plays a role in the biosynthesis of phosphorylcholine-derivatized LOS (Rock et al., J. Bacterol., 183: 4927-4931, 2001).

Also included among the in vivo-induced genes is a set whose products subsequently regulate gene expression or DNA replication. These genes include transcriptional regulation of glycerol metabolism by the glp repressor, glpR (SEQ ID NO: 643), the arginine repressor gene, argR (SEQ ID NO: 647), and the integration host factor (IHF) beta subunit, ihfB (SEQ ID NO: 645). IHF is a histone-like protein that binds DNA at specific sequences, an accessory factor involved in replication, site-specific recombination and transcription, altering the activity of a large number of operons (Goosen and van de Putte, Mol. Microbiol. 16: 1-7, 1995). In addition, CspD inhibits DNA replication during stationary phase-induced stress response in E. coli (Yamanaka et al., Mol. Microbiol., 39: 1572-1584, 2001) and the mukF (SEQ ID NO: 641) gene protein homologue contributes to a remodeling of the nucleiod structure into a more compact form prior to cell segregation (Sawitzke and Austin, Proc. Natl. Acad. Sci. U.S.A., 62: 1710-1718, 2000). The DFI strategy described herein also identified promoters induced in vivo for genes of unknown function. The hypothetical protein, HI0094, demonstrated an 8-fold increase in gene expression during early OM but its role remains unknown. HI1163 (SEQ ID NO: 651) showed 58% amino acid identity with the hypothetical YdiJ proteins, a putative oxidase, of E. coli.

A high-density transposon mutagenesis strategy was used to identify H. influenzae genes essential for growth on rich medium (Akerley et al., Proc. Natl. Acad. Sci. U.S.A., 99: 966-971, 2002). Six genes were identified in the screen described herein that are included in essential gene set described in Akerley' et al., supra. (hisB, lppB, lolA, ispZ, mukF and unknown HI0665). Recently genes of non-typeable H. influenzae that are expressed upon interaction with two human respiratory tract-derived epithelial cell lines have been identified. These genes included those involved in metabolic processes, stress responses, gene expression, cell envelope biosynthesis, DNA-related processes, cell division and ORF's encoding proteins of unknown function. (Ulsen et al., Mol. Microbiol., 45: 485-500, 2002). Similarly the stress response gene, cspD (SEQ ID NO: 649), genes involved in purine and riboflavin biosynthesis, and a protein of unknown function, vapA was identified in the screen described herein. Expression of vapA was detected in vitro, yet vapA gene expression increased two-fold in vivo. These unique approaches identified known genes that are upregulated in NTHi-induced OM and therefore are likely to play a role in NTHi infection and virulence; and may be potential candidates for vaccines and antisense therapies and other therapeutic methods of treatment of NTHi related disorders.

The DFI strategy resulted in the identification of promoters induced in vivo for genes of unknown function as well. The hypothetical protein, HI0094, demonstrated a 8-fold increase in gene expression during early OM but its role remains unknown. HI1163 (SEQ ID NO: 651) showed 58% amino acid identity with the hypothetical YdiJ proteins, a putative oxidase, of E. coli. Therefore, these hypothetical genes are likely to play a role in OM induced by NTHi infection.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 depicts the LKP gene region in a panel of Haemophilus isolates. The strain 86-028NP sequence is identical in this region to the sequence in NTHi strain R3001. Both of these NTHi lack the hif gene cluster encoding the hemagglutinating pilus.

FIG. 2 depicts the rfaD region in a panel of Haemophilus isolates. The gene arrangement in the rfaD region of the strain 86-028NP genome is similar to that of the strain Rd genome but different than the arrangement of these genes seen in the genome of most NTHi examined.

FIGS. 3A-3M set out the nucleotide sequences (SEQ ID NOS: 589-614) described in Table 4, which were identified to be upregulated during OM infection (see Example 6). The nucleotides (nt.) which correspond to known genes and those nt. which correspond to the contig sequences set out as SEQ ID NO: 1-576 are also presented.

DETAILED DESCRIPTION

The following examples illustrate the invention wherein Example 1 describes the sequence of the NTHi genome, Example 2 describes the identified contigs and initial gene discovery, Example 3 describes construction of the NTHi promoter trap library, Example 4 describes the analyses of 86-028NP derivatives expressing GFP, Example 5 demonstrates direct labelling of bacteria from middle ear fluids, Example 6 describes identification of promoters induced in vivo in acute otitis media, Example 7 describes identification of virulence-associated genes, Example 8 describes identification of unique NTHi gene sequences, Example 9 described the analysis of the complete NTHi 86-028NP genome, and Example 10 compares the genomic DNA sequences of NTHi, strain 86-028NP and H. influenzae serotype d, strain kw20

Example 1 Sequence of a Non-Typeable Haemophilus influenzae Genome

NTHi strain 86-028NP is a minimally passaged clinical isolate obtained from a pediatric patient who underwent tympanostomy and tube insertion for chronic OM at Columbus Children's Hospital. (Bakaletz et al. Infection and Immunity, 56(2): 331-335, 1988) The 86-028NP strain was deposited with the American Type Tissue Collection (Manassas, Va. 20108 USA) on Oct. 16, 2002 and assigned accession no. PTA-4764.

In an effort to more broadly approach the identification of the virulence determinants in NTHi, the genome of the NTHi 86-028NP strain was sequenced to 3-fold coverage. Chromosomal DNA was prepared from strain 86-028NP using the Puregene protocol and sheared to 2-4 kb in size with a Hydroshear instrument (Gene Machines). The sheared DNA was ethanol-precipitated, end-repaired using a mixture of Klenow enzyme and T4 DNA polymerase, and size-selected by agarose gel electrophoresis to obtain 2-4 kb fragments as described in Chissoe et al. (Methods: a Companion to Methods of Enzymology 3: 55-65, 1991) and Sambrook et al. (Molecular Cloning: a Laboratory Manual, 2^(nd) Ed. Cold Springs Harbor, N.Y., 1989). These fragments were cloned into vector pUC18 using the SmaI restriction site (phosphatase-treated) and transformed into E. coli XL-1 Blue, selecting for ampicillin resistance. Colonies that contain inserts were identified by blue/white screening on LB-Amp plates containing X-gal, and transferred into 96-deep well plates containing 1.5 ml of TB-Amp (TB=Terrific Broth) broth. The deep-well plate cultures were grown overnight (18-22 hours) at 37° C. Template preparation, sequencing and contig assembly were performed.

Automated template preparation was performed on the Beckman Biomek 2000 automated robotics workstation as described in Chissoe et al., (supra.) Briefly, each 96-deep well plate, containing the clones prepared above, was centrifuged to pellet the cells, the supernatant decanted, and the cells frozen (if necessary) at −20° C. Four 96-deep well blocks were placed on the Biomek table, and the liquid handling robot was used to prepare the template using an automated version of a typical SDS—NaOH lysis protocol as described in Chissoe et al., (supra.). The final ethanol-precipitated templates were each dissolved in 50 μl ddH₂O, and used for DNA sequencing.

Sequencing reactions were run by re-arraying the templates (from 96-well plates) into 384-well plates, using the Robbins Hydra 96 robot. Cycle-sequencing reactions were run using PE Big-Dye™ terminators and universal primers (M13 forward and reverse), cleaned up over Sephadex G50 columns, and analyzed on a PE Biosystems 3700 capillary electrophoresis DNA sequencer according to the manufacturer's instructions. Sequencing reads (8219) were assembled into 576 contigs (SEQ ID NOS: 1-576 herein). The statistics for the 3-fold sequencing are shown in Table 2A. The total unique sequence in assembly 17 is 1.74 Mb.

TABLE 2A Contig Size Total Number Total Length % of Cumulative 0-1 kb 65 55961  3.2% 1-2 kb 228 333665 19.2% 2-3 kb 101 243059 14.0% 3-4 kb 49 172385  9.9% 4-5 kb 45 196699 11.3% 5-10 kb 74 515152 29.6% 10-20 kb 11 144591  8.3% 20-30 kb 3 77352  4.4%

Subsequently, 8-fold sequencing analysis of the NTHi genome was carried out. The 8-fold sequencing assembled the NTHi genome into 11 contigs. Contigs 5, 8, 9, 10, 12-18 are denoted as SEQ ID NOS: 675-685 herein. The statistics for the 8-fold sequencing are shown in Table 2B.

TABLE 2B Contig Size Total Number Total Length % of Cumulative 0-1 kb 5 3950 0.2% 1-2 kb 3 4316 0.2% 2-3 kb 0 0 0.0% 3-4 kb 1 3964 0.2% 4-5 kb 0 0 0.0% 5-10 kb 0 0 0.0% 10-20 kb 1 15147 0.8% 20-30 kb 2 51888 2.7% 30-40 kb 0 0 0.0% 40-50 kb 0 0 0.0% 50-100 kb 1 85814 4.5% >100 kb 5 1760339 91.4% 

Example 2 Contig Description and Initial Gene Discovery

Seventy-five of the 88 contigs with length≧5000 bp, identified with the 3-fold sequence analysis, show significant similarity via BLASTN to genes in H. influenzae strain Rd. To visualize the potential relationship between the gene order in H. influenzae strain 86-028NP and H. influenzae strain Rd, the 86-028NP three-fold contig set and the Rd gene set were bidirectionally compared using BLASTN. The results were plotted in gene-order verses contig space by sorting the contigs based on gene coordinates of the Rd genes hit, anchoring each contig at the smallest coordinate found as described in Ray et al., (Bioinformatics 17: 1105-12, 2001). Compared in this fashion, an incomplete assembly of a genome with identical gene order to a completely known genome would display a monotonically increasing stair-stepped form.

BLASTX was used to identify hits to sequences with homology to genes in the strain Rd genome as well as genes not found in H. influenzae strain Rd. Hits to strain Rd sequences were removed from the data set and the other hits summarized in Table 3A. The data are presented as follows: contig # (SEQ ID NO: #), column 1; E score for each hit, column 2; the name of the protein that had homology to a portion of the amino acid translation of the cited contig, column 3; the organism producing the homologue, column 4; and the Genbank protein identifier for each of the proteins cited in column 3, column 5; the corresponding nucleotides within the contig (referenced by SEQ ID NO:). In most instances, several homologues were identified but for clarity, the protein of greatest homology is cited in Table 3A.

The sequences for some of the genes listed in Table 3A were identified within the 8-fold sequencing of the NTHi genome. Table 3B lists the location of these genes within the 11 contigs, the full length open reading frame sequence (identified by SEQ ID NO:), the derived amino acid sequence encoded by the open reading frame and the gene with high homology identified by BLASTX (as listed in Table 3A).

To examine the relative short range gene arrangements in NTHi and the Rd strain, the gene order in two gene clusters that have been well-described were compared. First, the genes present in the hemagglutinating pilus (LKP) gene region were examined. (Mhlanga-Mutangadura et al., J. Bacteriol. 180(17): 4693-703, 1998). The pilus gene cluster is located between the purE and pepN genes, only fragments of which are depicted in FIG. 1. The serotype b strain, Eagan, contains the hifABCDE gene cluster and produces hemagglutinating pili. Strain Rd lacks the hicAB genes as well as the hifABCDE gene cluster. In general, the nontypeable strains previously examined contained the hicAB genes but not the hif genes that encode the hemagglutinating pilus. The strain 86-028NP sequence (described herein) is identical in this region to the sequence in NTHi strain R3001 (FIG. 1). The rfaD gene encodes an enzyme involved in the biosynthesis of endotoxin. In addition, the rfaD gene from NTHi strain 2019 has been characterized by Nichols et al. (Infect Immunity 65(4): 1377-86, 1997). In strain 2019, the rfaD gene is immediately upstream of the rfaF gene that encodes another enzyme involved in endotoxin biosynthesis. The gene arrangement in strain Rd is different; the rfaD and rfaF genes are separated by approximately 11 kb of sequence. Most nontypeable strains examined contained the gene arrangement seen in strain 2019. In contrast, strain 86-028NP has a gene arrangement identical to that seen in strain Rd (FIG. 2).

A global analysis of the current assembly indicates that the gene content and order are similar to that in strain Rd. A more detailed analysis revealed that there are a substantial number of NTHi genes not previously seen in the Pasteurellaceae and some regions where the NTHi gene content and order is different than that seen in strain Rd. Thus, the current data suggest that the strain 86-028NP genome will contain a complex mosaic of Rd and non-Rd like features.

The DFI strategy also identified novel NTHi sequences that had increased gene expression. A list of these novel contig sequences that contain genes or gene fragments that have homology to ORFs in other organisms (primarily gram-negative bacteria) is set out in Table 3A. For example, the nucleotide sequence of contig 442 (SEQ ID NO: 442), nucleotides 1498-1845 are highly homologous to the sequences encoding amino acids 1-116 of H. influenzae strain Rd lipoprotein B (LppB). The gene is positioned between the stationary phase survival gene, surE, and a gene encoding a 43 kD antigenic outer membrane lipoprotein that is highly homologous to the recently identified bacterial lipoprotein, LppB/NlpD, which has been associated with virulence (Padmalayam et al., Infect. Immun., 68: 4972-4979, 2000). Recently, Zhang and coworkers demonstrated that nlpD and surE gene expression was induced during stationary phase of bacterial growth in Thermotoga maritima (Zhang et al., Structure (Camb), 9: 1095-1106, 2001). Therefore, under stress-induced conditions in the middle ear, this NTHi lipoprotein may be expressed.

TABLE 3A Genbank Contig E score Hit Identity Organism Protein SEQ ID NO: 104 4.00E−59 CpdB Pasteurella NP_246953.1 nt. 204-659 of multocida SEQ ID NO: 104 106 9.00E−10 hypothetical protein Pyrococcus G71244 nt. 40-309 of PH0217 - horikoshii SEQ ID NO: 106 106 1.00E−08 unknown Pasteurella NP_246871.1 nt. 605-694 of multocida SEQ ID NO: 106 106 2.00E−20 Orf122 Chlorobium AAG12204.1 nt. 7-210 of tepidum SEQ ID NO: 106 110 3.00E−05 ArdC antirestriction IncW plasmid pSa AAD52160.1 compliment of protein nt. 959-1162 of SEQ ID NO: 110 110 1.00E−33 hypothetical protein Salmonella NP_458676.1 compliment of enterica subsp. nt. nt. 181-825 of enterica serovar SEQ ID NO: 110 Typhi 111 5.00E−12 putative membrane Salmonella NP_458664.1 compliment of protein enterica subsp. nt. 45-287 of enterica serovar SEQ ID NO: 111 Typhi 111 6.00E−41 hypothetical protein Salmonella NP_458658.1 compliment of enterica subsp. nt. 1091-1480 of enterica serovar SEQ ID NO: 111 Typhi 114 7.00E−80 unknown Pasteurella NP_245828.1 compliment of multocida nt. 118-696 of SEQ ID NO: 114 115 2.00E−09 A111R Paramecium NP_048459.1 nt. 555-869 of bursaria Chlorella SEQ ID NO: 115 virus 1 118 5.00E−45 DNA methylase Vibrio cholerae NP_231404.1 nt. 44-439 of HsdM, putative SEQ ID NO: 118 122 2.00E−18 unknown Pasteurella NP_245314.1 nt. 865-1302 of multocida SEQ ID NO: 122 123 4.00E−99 RNA Proteus mirabilis P50509 nt. 351-782 of POLYMERASE SEQ ID NO: 123 SIGMA-32 FACTOR 124 9.00E−58 ACETOLACTATE Spirulina platensis P27868 nt. 603-1025 of SYNTHASE SEQ ID NO: 124 (ACETOHYDROXY- ACID SYNTHASE) (ALS) 130 0 restriction Neisseria CAA09003.1 nt. 495-1559 of modification meningitidis SEQ ID NO: 130 system-R protein 131 6.00E−91 uronate isomerase Salmonella NP_457532.1 compliment of (glucuronate enterica subsp. nt. 661-1380 of isomerase) enterica serovar SEQ ID NO: 131 Typhi 133 3.00E−30 GyrA Pasteurella NP_245778.1 compliment of multocida nt. 1447-1626 of SEQ ID NO: 133 133 1.00E−27 DNA GYRASE Pectobacterium P41513 compliment of SUBUNIT A carotovorum nt. 1302-1442 of SEQ ID NO: 133 138 7.00E−06 KicA Pasteurella NP_245545.1 compliment of multocida nt. 92-157 of SEQ ID NO: 138 138  1.00E−148 TYPE II Haemophilus O30869 compliment of RESTRICTION aegyptius nt. 164-1045 of ENZYME HAEII SEQ ID NO: 138 (ENDONUCLEASE HAEII) (R. HAEII) 143 4.00E−06 Gifsy-1 prophage Salmonella NP_461555.1 compliment of protein typhimurium LT2 nt. 228-632 of SEQ ID NO: 143 143 1.00E−14 hypothetical protein Bacteriophage NP_050531.1 compliment of VT2-Sa nt. 778-1248 of SEQ ID NO: 143 143 5.00E−09 hypothetical protein Salmonella CAD09979.1 compliment of enterica subsp. nt. 715-1026 of enterica serovar SEQ ID NO: 143 Typhi 143 6.00E−10 hypothetical 14.9 kd Escherichia coli NP_065324.1 nt. 3-173 of protein SEQ ID NO: 143 147 1.00E−38 GTP-binding Escherichia coli NP_289127.1 compliment of elongation factor, O157:H7 EDL933 nt. 172-342 of may be inner SEQ ID NO: 147 membrane protein 147 2.00E−14 GTP-binding Borrelia NP_212222.1 compliment of membrane protein burgdorferi nt. 17-181 of (lepA) SEQ ID NO: 147 148 6.00E−17 galactokinase Homo sapiens AAC35849.1 compliment of nt. 746-1246 of SEQ ID NO: 148 148 7.00E−96 GALACTOKINASE Actinobacillus P94169 compliment of (GALACTOSE pleuropneumoniae nt. 232-741 of KINASE) SEQ ID NO: 148 149 1.00E−92 GTP-binding Buchnera sp. NP_240245.1 compliment of protein TypA/BipA APS nt. 265-1077 of SEQ ID NO: 149 15 2.00E−21 ORF 1 Escherichia coli CAA39631.1 nt: 665-850 of SEQ ID NO: 15 150 6.00E−17 unknown Pasteurella NP_245919.1 nt. 171-665 of multocida SEQ ID NO: 150 153 7.00E−07 outer membrane Rickettsia conorii T30852 nt. 51-623 of protein A SEQ ID NO: 153 155 7.00E−40 cytochrome d Vibrio cholerae NP_233259.1 nt. 583-1002 of ubiquinol oxidase, SEQ ID NO: 155 subunit II 157 7.00E−13 unknown Pasteurella NP_245490.1 compliment of multocida nt. 1170-1367 of SEQ ID NO: 157 157 2.00E−05 glycosyl Neisseria AAA68012.1 nt. 85-189 of transferase gonorrhoeae SEQ ID NO: 157 158  1.00E−152 MltC Pasteurella NP_246259.1 compliment of multocida nt. 36-530 of SEQ ID NO: 158 161 3.00E−25 lipoprotein, putative Vibrio cholerae NP_230232.1 nt. 870-1439 of SEQ ID NO: 161 163 9.00E−53 chorismate Caulobacter NP_421948.1 nt. 1283-2029 of synthase crescentus SEQ ID NO: 163 168 3.00E−13 COPPER- Mus musculus Q64430 nt. 66-995 of TRANSPORTING SEQ ID NO: 168 ATPASE 1 (COPPER PUMP 1) 168 2.00E−22 Cu transporting Homo sapiens 2001422A nt. 135-989 of ATPase P SEQ ID NO: 168 174 8.00E−48 magnesium/cobalt Mesorhizobium NP_103977.1 nt. 918-1205 of transport protein loti SEQ ID NO: 174 175 5.00E−26 vacB protein Buchnera sp. NP_240369.1 compliment of APS nt. 1-1587 of SEQ ID NO: 175 176 3.00E−21 putative ABC Campylobacter NP_282774.1 compliment of transport system jejuni nt. 259-1089 of permease protein [ SEQ ID NO: 176 183 5.00E−29 PROBABLE ATP Ralstonia NP_521442.1 compliment of SYNTHASE A solanacearum nt. 42-677 of CHAIN SEQ ID NO: 183 TRANSMEMBRANE PROTEIN 185 6.00E−85 putative exported Salmonella NP_458655.1 compliment of protein enterica subsp. nt. 162-1529 of enterica serovar SEQ ID NO: 185 Typhi 187 2.00E−05 transketolase Homo sapiens AAA61222.1 nt. 709-819 of SEQ ID NO: 187 188  1.00E−116 ribonuclease E Xylella fastidiosa NP_299884.1 compliment of 9a5c nt. 280-1704 of SEQ ID NO: 188 192 1.00E−38 ImpA Pasteurella NP_245829.1 nt. 35-448 of multocida SEQ ID NO: 192 193 3.00E−08 Orf80 Enterobacteria NP_052285.1 nt. 1612-1818 of phage 186 SEQ ID NO: 193 193 1.00E−06 holin Haemophilus AAC45168.1 nt. 370-576 of somnus SEQ ID NO: 193 193    0.007 unknown Enterobacteria NP_052260.1 nt. 1376-1609 of phage 186 SEQ ID NO: 193 193 2.00E−48 lysozyme Haemophilus AAC45169.1 nt. 608-1093 of somnus SEQ ID NO: 193 199 4.00E−21 unknown protein Escherichia coli NP_288675.1 nt. 398-778 of O157:H7 SEQ ID NO: 199 EDL933, prophage CP- 933V 199 2.00E−49 hypothetical protein Bacteriophage NP_049495.1 compliment of 933W nt. 1907-2392 of SEQ ID NO: 199 20 1.00E−62 RpL14 Pasteurella NP_246344.1 compliment of multocida nt. 233-601 of SEQ ID NO: 20 200 2.00E−62 hypothetical protein Salmonella NP_458658.1 compliment of enterica subsp. nt. 431-997 of enterica serovar SEQ ID NO: 200 Typhi 200 3.00E−16 hypothetical protein Salmonella NP_458657.1 compliment of enterica subsp. nt. 1028-1264 of enterica serovar SEQ ID NO: 200 Typhi 201 2.00E−26 TsaA Pasteurella NP_245732.1 compliment of multocida nt. 1618-1809 of SEQ ID NO: 201 209 6.00E−16 TsaA Pasteurella NP_245732.1 compliment of multocida nt. 2-136 of SEQ ID NO: 209 211 2.00E−15 unknown Pasteurella NP_245535.1 compliment of multocida nt. 23-211 of SEQ ID NO: 211 211 1.00E−70 PUTATIVE Ralstonia NP_520082.1 compliment of ATPASE PROTEIN solanacearum nt. 475-915 of SEQ ID NO: 211 212 3.00E−18 hypothetical protein Escherichia coli NP_309775.1 compliment of O157:H7 nt. 895-1035 of SEQ ID NO: 212 216  1.00E−173 unknown Pasteurella NP_245069.1 nt. 35-1543 of multocida SEQ ID NO: 216 217 9.00E−18 diacylglycerol Vibrio cholerae NP_233101.1 nt. 2083-2208 of kinase SEQ ID NO: 217 221 4.00E−34 Tail-Specific Chlamydia NP_219953.1 nt. 849-1421 of Protease trachomatis SEQ ID NO: 221 222 4.00E−23 AGR_C_3689p Agrobacterium NP_355005.1 compliment of tumefaciens str. nt. 940-1305 of C58 (Cereon) SEQ ID NO: 222 224 9.00E−19 unknown Pasteurella NP_245536.1 nt. 15-308 of multocida SEQ ID NO: 224 225 1.00E−89 portal vector -like Salmonella NP_461651.1 nt. 31-750 of of protein, in phage typhimurium SEQ ID NO: 225 P2 [Salmonella LT2Fels-2 typhimurium LT2] prophage 229 2.00E−25 anaerobic Salmonella CAB62266.1 nt. 1806-2108 of ribonucleotide typhimurium SEQ ID NO: 229 reductase 234 3.00E−08 conserved Xylella fastidiosa NP_299850.1 nt. 1680-2048 of hypothetical protein 9a5c SEQ ID NO: 234 234 1.00E−42 Methionine Clostridium NP_348177.1 compliment of sulfoxide reductase acetobutylicum nt. 415-654 of C-terminal domain SEQ ID NO: 234 related protein, YPPQ ortholog 235 4.00E−16 phage-related tail Wolbachia AAK85310.1 compliment of protein endosymbiont of nt. 931-1929 of Drosophila SEQ ID NO: 235 melanogaster 235 6.00E−56 similar to orfG Salmonella NP_461625.1 compliment of protein in phage typhimurium LT2, nt. 313-1863 of 186, Salmonella Fels-2 prophage SEQ ID NO: 235 typhimurium LT2 236 6.00E−20 conserved Pseudomonas NP_252693.1 nt. 1572-1916 of hypothetical protein aeruginosa SEQ ID NO: 236 240 5.00E−27 MODIFICATION Brevibacterium P10283 compliment of METHYLASE BEPI epidermidis nt. 922-1305 of SEQ ID NO: 240 241 2.00E−15 phage-related Xylella fastidiosa NP_299573.1 compliment of protein 9a5c nt. 865-1305 of SEQ ID NO: 241 241 4.00E−08 hypothetical protein phage SPP1 T42296 nt. 73-636 of SEQ ID NO: 241 241 4.00E−07 hypothetical protein Salmonella NP_458686.1 nt. 10-468 of enterica subsp. SEQ ID NO: 241 enterica serovar Typhi 242 2.00E−29 translation chloroplast - S35701 compliment of elongation factor soybean nt. 18-1085 of EF-G SEQ ID NO: 242 247 3.00E−23 GTP Synechococcus Q54769 compliment of CYCLOHYDROLA sp. PCC 7942 nt. 1009-1257c of SE I (GTP-CH-I) SEQ ID NO: 247 248 6.00E−05 phospho-N- Aquifex aeolicus NP_213025.1 nt. 830-1747 of acetylmuramoyl- SEQ ID NO: 248 pentapeptide- transferase 25 2.00E−86 PROBABLE Ralstonia NP_522358.1 compliment of TRANSPORT solanacearum nt. 309-854 of TRANSMEMBRANE SEQ ID NO: 25 PROTEIN 25 7.00E−06 major facilitator Caulobacter NP_419155.1 compliment of family transporter crescentus nt. 134-283 of SEQ ID NO: 25 250  1.00E−150 CpdB Pasteurella NP_246953.1 compliment of multocida nt. 36-1016 of SEQ ID NO: 250 252 3.00E−57 alanyl-tRNA Vibrio cholerae AAA99922.1 compliment of synthetase nt. 1418-1951 of SEQ ID NO: 252 253  1.00E−108 similar to Listeria NP_464432.1 compliment of glutathione monocytogenes nt. 411-1358 of of Reductase EGD-e SEQ ID NO: 253 259 3.00E−39 hypothetical protein Salmonella NP_458654.1 compliment of enterica subsp. nt. 342-1037 of enterica serovar SEQ ID NO: 259 Typhi 259 3.00E−17 possible exported Salmonella NP_458653.1 compliment of protein enterica subsp. nt. 1251-1607 of enterica serovar SEQ ID NO: 259 Typhi 261 5.00E−74 hypothetical protein Haemophilus S27582 compliment of 6 - Haemophilus influenzae nt. 3-422 of influenzae SEQ ID NO: 261 263 1.00E−94 putative Haemophilus AAD01406.1 nt. 2142-2672 of transposase paragallinarum SEQ ID NO: 263 264  1.00E−126 unknown Actinobacillus NP_067554.1 nt. 40-714 of actinomycetemco- SEQ ID NO: 264 mitans 264  1.00E−103 unknown Actinobacillus NP_067555.1 nt. 695-1309 of actinomycetemco- SEQ ID NO: 264 mitans 264 2.00E−21 unknown Actinobacillus NP_067556.1 nt. 1302-1448 of actinomycetemco- SEQ ID NO: 264 mitans 265 6.00E−27 Aminopeptidase 2 chloroplast Q42876 nt. 556-1539 of SEQ ID NO: 265 268  1.00E−116 MutY Pasteurella NP_246257.1 nt. 1003-1581 of multocida SEQ ID NO: 268 272 1.00E−07 hypothetical protein Bacteriophage NP_049495.1 compliment of 933W nt. 77-232 of SEQ ID NO: 272 274 3.00E−13 unknown Pasteurella NP_246952.1 compliment of multocida nt. 1658-1975 of SEQ ID NO: 274 275 3.00E−20 CafA Neisseria AAG24267.1 nt. 1299-1571 of gonorrhoeae SEQ ID NO: 275 276 1.00E−45 mukE protein Vibrio cholerae NP_231351.1 compliment of nt. 650-1390 of SEQ ID NO: 276 276 1.00E−69 KicA Pasteurella NP_245545.1 compliment of multocida nt. 647-1321 of SEQ ID NO: 276 278 2.00E−56 3-oxoacyl-[acyl- Salmonella NP_455686.1 nt. 1366-1944 of carrier-protein] enterica subsp. SEQ ID NO: 278 synthase III enterica serovar Typhi 281 5.00E−56 unknown Pasteurella NP_246261.1 compliment of multocida nt. 31-678 of SEQ ID NO: 281 282 3.00E−09 orf25; similar to T bacteriophage phi NP_490625.1 compliment of gene of P2 CTX nt. 511-1032 of SEQ ID NO: 282 282 1.00E−08 orf11; similar to Haemophilus AAC45165.1 compliment of phage P2 gene S- somnus nt. 1450-1584 of like product, which SEQ ID NO: 282 is involved in tail synthesis, 282 9.00E−27 putative Salmonella NP_457167.1 compliment of bacteriophage tail enterica subsp. nt. 3-509 of protein enterica serovar SEQ ID NO: 282 Typhi 286 5.00E−18 plasmid-related Listeria innocua NP_471066.1 compliment of protein plasmid nt. 887-1501 of SEQ ID NO: 286 287 8.00E−20 GTP Escherichia coli NP_287920.1 nt. 2-145 of cyclohydrolase II O157:H7 EDL933 SEQ ID NO: 287 289  1.00E−168 MODIFICATION Haemophilus O30868 compliment of METHYLASE aegyptius nt. 138-1091 of HAEII SEQ ID NO: 289 289 5.00E−11 TYPE II Haemophilus O30869 compliment of RESTRICTION aegyptius nt. 22-132 of ENZYME HAEII SEQ ID NO: 289 289 6.00E−47 mukF homolog Haemophilus AAB70828.1 compliment of influenzae biotype nt. 1107-1385 of aegyptius SEQ ID NO: 289 294  1.00E−171 LICA PROTEIN Haemophilus P14181 compliment of influenzae nt. 677-1564 of RM7004 SEQ ID NO: 294 297  1.00E−158 DNA methylase Vibrio cholerae NP_231404.1 compliment of HsdM, putative nt. 12-1136 of SEQ ID NO: 297 302 0 HEME-BINDING Haemophilus P33950 nt. 3-1316 of PROTEIN A influenzae DL42 SEQ ID NO: 302 304 6.00E−19 hypothetical protein Haemophilus S27582 nt. 121-267 of 6 influenzae SEQ ID NO: 304 305 6.00E−40 putative Streptococcus NP_269557.1 nt. 65-805 of recombinase - pyogenes M1 SEQ ID NO: 305 phage associated GAS 305 7.00E−22 single stranded Shewanella sp. AAB57886.1 nt. 1607-2014 of DNA-binding F1A SEQ ID NO: 305 protein 305 1.00E−43 phage-related Bacillus NP_244410.1 nt. 92-751 of protein halodurans SEQ ID NO: 305 312 1.00E−28 PUTATIVE Ralstonia NP_518994.1 nt. 1819-2673 of BACTERIOPHAGE- solanacearum SEQ ID NO: 312 RELATED TRANSMEMBRANE PROTEIN 312 9.00E−25 similar to Homo sapiens XP_068727.1 nt. 27-1001 of BASEMENT SEQ ID NO: 312 MEMBRANE- SPECIFIC HEPARAN SULFATE PROTEOGLYCAN CORE PROTEIN PRECURSOR (HSPG) 315 2.00E−45 uracil permease Deinococcus NP_296001.1 compliment of radiodurans nt. 525-1592 of SEQ ID NO: 315 318 7.00E−15 CzcD Pasteurella NP_246276.1 compliment of multocida nt. 3-227 of SEQ ID NO: 318 320 2.00E−60 orf3; similar to Haemophilus AAC45159.1 compliment of endonuclease somnus nt. 606-1241 of subunit of the SEQ ID NO: 320 phage P2 terminase (gene M) 320 2.00E−09 orf4; similar to Haemophilus AAC45160.1 compliment of head somnus nt. 52-285 of completion/stabili- SEQ ID NO: 320 zation protein (gene L) of phage P2 320 3.00E−35 orf2; similar to Haemophilus AAC45158.1 compliment of major capsid somnus nt. 1271-1624 of protein precursor of SEQ ID NO: 320 phage P2 (gene N) 323 4.00E−37 dedC protein Escherichia coli AAA23966.1 compliment of nt. 74-463 of SEQ ID NO: 323 324  1.00E−153 conserved Neisseria NP_274972.1 compliment of hypothetical protein meningitidis nt. 930-1943 of MC58 SEQ ID NO: 324 326 5.00E−52 selenophosphate Eubacterium CAB53511.1 compliment of synthetase acidaminophilum nt. 1186-2292 of SEQ ID NO: 326 328  1.00E−129 secretion protein Pseudomonas NP_252510.1 compliment of SecD aeruginosa nt. 8-625 of SEQ ID NO: 328 333 3.00E−08 unknown Pasteurella NP_245489.1 compliment of multocida nt. 5-418 of SEQ ID NO: 333 336 6.00E−38 probable methyl Pseudomonas NP_253353.1 compliment of transferase aeruginosa nt. 2547-2819 of SEQ ID NO: 336 338 2.00E−98 Pmi Pasteurella NP_245766.1 nt. 144-842 of multocida SEQ ID NO: 338 339 2.00E−07 tRNA Escherichia coli QQECPE nt. 2331-2540 of nucleotidyltransferase SEQ ID NO: 339 340 0 DNA gyrase, Salmonella NP_461214.1 compliment of subunit A, type II typhimurium LT2 nt. 93-1799 of topoisomerase SEQ ID NO: 340 342 4.00E−12 tolA protein Haemophilus JC5212 nt. 980-1318 of influenzae SEQ ID NO: 342 344 1.00E−07 MODIFICATION Haemophilus P50192 compliment of METHYLASE parahaemolyticus nt. 849-1034 of HPHIA SEQ ID NO: 344 344 8.00E−05 ABC transporter Leishmania major AAF31030.1 compliment of protein 1 nt. 17-205 of SEQ ID NO: 344 349 3.00E−44 conserved Neisseria NP_273467.1 compliment of hypothetical protein meningitidis nt. 1397-1903 of MC58 SEQ ID NO: 349 349 8.00E−09 hypothetical protein Pseudomonas NP_252667.1 compliment of aeruginosa nt. 795-1121 of SEQ ID NO: 349 349 9.00E−10 conserved Helicobacter NP_207009.1 compliment of hypothetical pylori 26695 nt. 1319-1816 of secreted protein SEQ ID NO: 349 349 2.00E−06 putative TPR Salmonella NP_463149.1 compliment of repeat protein typhimurium LT2 nt. 2244-2558 of SEQ ID NO: 349 35 1.00E−23 type I restriction- Xylella fastidiosa NP_300003.1 compliment of modification 9a5c nt. 29-388 of system specificity SEQ ID NO: 35 determinant 352  1.00E−116 putative peptidase Escherichia coli NP_416827.1 compliment of K12 nt. 951-1640 of SEQ ID NO: 352 352 0 unknown Pasteurella NP_245275.1 compliment of multocida nt. 86-946 of SEQ ID NO: 352 354 5.00E−86 putative uronate Salmonella NP_462052.1 compliment of isomerase typhimurium LT2 nt. 168-914 of SEQ ID NO: 354 356 1.00E−07 isomerase-like Escherichia coli S57220 nt. 5-73 of protein (DsbD) - SEQ ID NO: 356 358 1.00E−07 USG protein Pediococcus CAC16793.1 nt. 534-1307 of pentosaceus SEQ ID NO: 358 358 0.005 HsdS protein Escherichia coli CAA10700.1 nt. 26-205 of SEQ ID NO: 358 361  1.00E−152 maltodextrin Escherichia coli NP_289957.1 compliment of phosphorylase O157:H7 EDL933 nt. 77-922 of SEQ ID NO: 361 363 6.00E−06 BH2505-unknown Bacillus NP_243371.1 nt. 554-844 of conserved protein halodurans SEQ ID NO: 363 368 1.00E−12 H02F09.3.p Caenorhabditis NP_508295.1 compliment of elegans nt. 1069-1977 of SEQ ID NO: 368 368 6.00E−27 hypothetical Mesorhizobium NP_102360.1 compliment of glycine-rich protein loti nt. 1201-1986 of SEQ ID NO: 368 37 6.00E−09 putative ATP- Escherichia coli NP_415469.1 compliment of binding component K12 nt. 455-691 of of a transport SEQ ID NO: 37 system 372 7.00E−18 conserved Clostridium BAB80319.1 compliment of hypothetical protein perfringens nt. 1763-1924 of SEQ ID NO: 372 376 7.00E−24 putative Salmonella NP_456379.1 compliment of bacteriophage enterica subsp. nt. 158-808 of protein enterica serovar SEQ ID NO: 376 Typhi 376 8.00E−10 hypothetical protein Xylella fastidiosa NP_298882.1 compliment of 9a5c nt. 1129-1671 of SEQ ID NO: 376 376 9.00E−06 Iin1713 Listeria innocua NP_471049.1 compliment of nt. 913-1557 of SEQ ID NO: 376 377 6.00E−05 Vng1732c Halobacterium sp. NP_260487.1 nt. 2378-2587 of NRC-1 SEQ ID NO: 377 377 1.00E−11 INVASIN Yersinia P31489 compliment of PRECURSOR enterocolitica nt. 127-345 of (OUTER SEQ ID NO: 377 MEMBRANE ADHESIN) 382 4.00E−16 unknown Pasteurella NP_246871.1 compliment of multocida nt. 967-1068 of SEQ ID NO: 382 383 4.00E−36 putative Streptomyces BAB69302.1 nt. 488-1162 of transposase avermitilis SEQ ID NO: 383 383 3.00E−58 recombinase IncN plasmid R46 NP_511241.1 compliment of nt. 1-393 of SEQ ID NO: 383 383 4.00E−24 transposase Escherichia coli I69674 nt. 1294-1740 of SEQ ID NO: 383 383 0 tnpA Yersinia CAA73750.1 nt. 1782-2834 of enterocolitica SEQ ID NO: 383 385 2.00E−31 unknown Pasteurella NP_246065.1 nt. 1515-1772 of multocida SEQ ID NO: 385 386 5.00E−65 cydC [ Escherichia coli AAA66172.1 compliment of nt. 3438-4115 of SEQ ID NO: 386 386 4.00E−33 ABC transporter, Mesorhizobium NP_105463.1 compliment of ATP-binding loti nt. 2569-3390 of protein SEQ ID NO: 386 388 3.00E−45 60 KDA INNER- Coxiella burnetii P45650 compliment of MEMBRANE nt. 3211-3759 of PROTEIN SEQ ID NO: 388 HOMOLOG 390 4.00E−25 putative DNA- Salmonella NP_458175.1 nt. 1051-1416 of binding protein enterica subsp. SEQ ID NO: 390 enterica serovar Typhi 390 3.00E−13 transcriptional Bacillus NP_241773.1 compliment of regulator halodurans nt. 84-578 of SEQ ID NO: 390 390 3.00E−06 DNA translocase Staphylocoecus NP_372265.1 compliment of stage III sporulation aureus subsp. nt. 620-871 of prot homolog aureus Mu50 SEQ ID NO: 390 395 7.00E−31 ATPase, Cu++ Homo sapiens NP_000044.1 compliment of transporting, beta nt. 615-1406 of polypeptide SEQ ID NO: 395 397 3.00E−23 terminase large Bacteriophage NP_112076.1 compliment of subunit HK620 nt. 2363-2725 of SEQ ID NO: 397 397 3.00E−16 hypothetical protein Xylella fastidiosa NP_297824.1 compliment of 9a5c nt. 1517-1744 of SEQ ID NO: 397 398 4.00E−67 orf32 Haemophiius NP_536839.1 compliment of phage HP2 nt. 1288-1866 of SEQ ID NO: 398 398 8.00E−24 putative Salmonella NP_463063.1 compliment of cytoplasmic protein typhimurium LT2 nt. 798-1220 of SEQ ID NO: 398 398 2.00E−83 orf31 Haemophilus NP_043502.1 compliment of phage HP1 nt. 1881-2510 of SEQ ID NO: 398 399 1.00E−94 HEME/HEMOPEXIN- Haemophilus P45355 nt. 88-774 of BINDING influenzae N182 SEQ ID NO: 399 PROTEIN 401 3.00E−63 Sty SBLI Salmonella CAA68058.1 nt. 1690-2742 of enterica SEQ ID NO: 401 401 3.00E−06 RESTRICTION- Mycoplasma NP_325912.1 nt. 79-489 of MODIFICATION pulmonis SEQ ID NO: 401 ENZYME SUBUNIT M3 402 2.00E−13 OPACITY Neisseria Q05033 compliment of PROTEIN OPA66 gonorrhoeae nt. 2634-2915 of PRECURSOR SEQ ID NO: 402 406 8.00E−13 type I restriction Neisseria NP_273876.1 nt. 281-520 of enzyme EcoR124IIR meningitidis SEQ ID NO: 406 MC58 407 6.00E−65 unknown Pasteurella NP_246237.1 nt. 938-2450 of multocida SEQ ID NO: 407 407 5.00E−99 PepE Pasteurella NP_245391.1 nt. 1216-1917 of multocida SEQ ID NO: 407 407 1.00E−16 Hemoglobin- Haemophilus Q48153 nt. 1-141 of haptoglobin binding influenzae Tn106 SEQ ID NO: 407 protein A 409  1.00E−106 hypothetical protein Haemophilus S27577 compliment of 1 influenzae nt. 2524-3159 of SEQ ID NO: 409 411 4.00E−29 heme-repressible Haemophilus AAB46794.1 nt. 391-615 of hemoglobin-binding influenzae, type b, SEQ ID NO: 411 protein strain HI689 411 0 Hemoglobin- Haemophilus Q48153 nt. 651-3263 of haptoglobin binding influenzae Tn106 SEQ ID NO: 411 protein A 412 2.00E−07 REGULATORY bacteriophage P03036 compliment of PROTEIN CRO 434 nt. 59-259 of (ANTIREPRESSOR) SEQ ID NO: 412 412 4.00E−06 hypothetical protein Bacteriophage CAC83535.1 nt. 1436-1654 of P27 SEQ ID NO: 412 413 8.00E−07 hypothetical protein Deinococcus NP_294301.1 compliment of radiodurans nt. 791-1012 of SEQ ID NO: 413 414 9.00E−65 conserved Vibrio cholerae NP_230092.1 nt. 1696-2103 of hypothetical protein SEQ ID NO: 414 414 3.00E−93 unknown Pasteurella NP_246834.1 nt. 1777-2109 of multocida SEQ ID NO: 414 416 2.00E−17 unknown Pasteurella NP_246629.1 compliment of multocida nt. 2565-2831 of SEQ ID NO: 416 416 4.00E−26 hypothetical protein Escherichia coli S30728 compliment of o154 nt. 1928-2254 of SEQ ID NO: 416 416 3.00E−37 transport protein Pseudomonas NP_253757.1 compliment of TatC aeruginosa nt. 1494-2018 of of SEQ ID NO: 416 417 1.00E−66 weakly similar to Listeria innocua NP_471073.1 compliment of methyltransferases nt. 999-1928 of SEQ ID NO: 417 417 5.00E−05 DNA-BINDING Pectobacterium Q47587 compliment of PROTEIN RDGA carotovorum nt. 3526-4212 of SEQ ID NO: 417 417 2.00E−29 putative phage- Yersinia pestis NP_407132.1 compliment of related protein nt. 2546-2938 of SEQ ID NO: 417 417 3.00E−06 Adenine-specific Thermoplasma NP_393798.1 compliment of DNA methylase acidophilum nt. 826-1020 of SEQ ID NO: 417 43 9.00E−16 PcnB Pasteurella NP_245801.1 nt. 511-870 of multocida SEQ ID NO: 43 434 2.00E−97 beta′ subunit of Nephroselmis NP_050840.1 compliment of RNA polymerase olivacea nt. 32-1534 of SEQ ID NO: 434 435 4.00E−52 MODIFICATION Brevibacterium P10283 compliment of METHYLASE BEPI epidermidis nt. 11-565 of SEQ ID NO: 435 435 4.00E−57 pentafunctional Saccharomyces NP_010412.1 compliment of arom polypeptide cerevisiae nt. 757-2064 of (contains: 3- SEQ ID NO: 435 dehydroquinate synthase, 3- dehydroquinate, dehydratase (3- dehydroquinase), shikimate 5- dehydrogenase, shikimate kinase, and epsp synthase) 437 5.00E−70 dihydrofolate Haemophilus S52336 nt. 2393-2767 of reductase influenzae SEQ ID NO: 437 (clinical isolate R1042) 438  1.00E−106 polyA polymerase Vibrio cholerae NP_230244.1 nt. 3-1124 of SEQ ID NO: 438 439 6.00E−60 Porphyrin Salmonella NP_457816.1 nt. 2343-2783 of biosynthetic protein enterica subsp. SEQ ID NO: 439 enterica serovar Typhi 441 5.00E−73 RimM Pasteurella NP_246234.1 compliment of multocida nt. 151-441 of SEQ ID NO: 441 442 9.00E−31 LIPOPROTEIN Salmonella P40827 compliment of NLPD typhimurium nt. 3362-3520 of SEQ ID NO: 442 444 6.00E−24 glycine betaine Staphylococcus NP_371872.1 compliment of transporter aureus subsp. nt. 2242-2514 of aureus Mu50 SEQ ID NO: 444 452 6.00E−28 unknown Pasteurella NP_245616.1 compliment of multocida nt. 533-883 of SEQ ID NO: 452 452 0 Type I restriction Escherichia coli Q47163 nt. 3291-4154 of enzyme Ecoprrl M SEQ ID NO: 452 protein 452 2.00E−75 type I restriction Ureaplasma NP_077929.1 nt. 4156-4662 of enzyme M protein urealyticum SEQ ID NO: 452 455 9.00E−56 PROBABLE Ralstonia NP_520059.1 nt. 2028-2774 of BACTERIOPHAGE solanacearum SEQ ID NO: 455 PROTEIN 455 2.00E−55 orf2; similar to Haemophilus AAC45158.1 nt. 2864-3490 of major capsid somnus SEQ ID NO: 455 protein precursor of phage P2 (gene N), 455  1.00E−175 gpP Enterobacteria NP_046758.1 compliment of phage P2 nt. 127-1812 of SEQ ID NO: 455 456 1.00E−38 hypothetical protein Pseudomonas NP_542872.1 compliment of putida nt. 1010-1282 of SEQ ID NO: 456 456  1.00E−172 hypothetical protein Pseudomonas NP_542873.1 compliment of putida nt. 1443-2006 of SEQ ID NO: 546 457  1.00E−116 hypothetical protein Haemophilus S15287 compliment of (galE 5′ region) - influenzae nt. 62-961 of Haemophilus SEQ ID NO: 457 influenzae 457  1.00E−134 dTDPglucose 4,6- Actinobacillus T00102 nt. 2637-3656 of dehydratase actinomycetemco- SEQ ID NO: 457 mitans 459 2.00E−10 RNA polymerase Synechocystis sp. NP_441586.1 nt. 25-117 of gamma-subunit PCC 6803 SEQ ID NO: 459 461 9.00E−51 conserved Staphylococcus NP_370593.1 nt. 4124-4624 of hypothetical protein aureus subsp. SEQ ID NO: 461 aureus Mu50 462 9.00E−06 NADH Burkholderia AAG01016.1 nt. 703-828 of dehydrogenase pseudomallei SEQ ID NO: 462 465 3.00E−41 GTP-binding Synechocystis sp. NP_441951.1 compliment of protein Era PCC 6803 nt. 2470-2787 of SEQ ID NO: 465 466 1.00E−15 putative Salmonella NP_455548.1 nt. 837-1478 of bacteriophage enterica subsp. SEQ ID NO: 466 protein enterica serovar Typhi 466 1.00E−90 orf31 Haemophilus NP_043502.1 nt. 2396-3199 of phage HP1 SEQ ID NO: 466 469 0 Hemoglobin and Haemophilus Q9X442 compliment of hemoglobin- influenzae HI689 nt. 427-3459 of haptoglobin binding SEQ ID NO: 469 protein C precursor 471 8.00E−05 transposase, Neisseria NP_274608.1 nt. 2957-3217 of putative meningitidis SEQ ID NO: 471 MC58 472 6.00E−08 hypothetical protein Salmonella NP_458660.1 compliment of enterica subsp. nt. 2881-3270 of enterica serovar SEQ ID NO: 472 Typhi 472 5.00E−23 antirestriction Mesorhizobium NP_106707.1 nt. 4908-5324 of protein loti SEQ ID NO: 472 472 1.00E−75 hypothetical protein Salmonella NP_458661.1 compliment of enterica subsp. nt. 1931-2776 of enterica serovar SEQ ID NO: 472 Typhi 472 9.00E−72 hypothetical protein Salmonella NP_458662.1 compliment of enterica subsp. nt. 544-1689 of enterica serovar SEQ ID NO: 472 Typhi 475 3.00E−25 unknown Pasteurella NP_244952.1 nt. 3207-3626 of multocida SEQ ID NO: 475 476 8.00E−73 putative DNA- Salmonella NP_458175.1 compliment of binding protein enterica subsp. nt. 3339-4310 of enterica serovar SEQ ID NO: 476 Typhi 476 6.00E−47 anticodon nuclease Neisseria NP_273873.1| compliment of meningitidis nt. 4397-4885 of MC58 SEQ ID NO: 476 478 3.00E−06 methionin Arabidopsis CAB38313.1 compliment of synthase-like thaliana nt. 3554-3679 of enzyme SEQ ID NO: 478 478 3.00E−05 unknown Pasteurella NP_245444.1 compliment of multocida nt. 164-250 of SEQ ID NO: 478 479 1.00E−18 conserved Xylella fastidiosa NP_298841.1 nt. 2302-2658 of hypothetical protein 9a5c SEQ ID NO: 479 48 3.00E−19 Dca Neisseria AAF12796.1 compliment of gonorrhoeae nt. 225-746 of SEQ ID NO: 48 482 1.00E−06 hypothetical protein Neisseria NP_275122.1 nt. 2055-2189 of meningitidis SEQ ID NO: 482 MC58 482 9.00E−28 conserved Neisseria NP_274383.1 nt. 1689-1898 of hypothetical protein meningitidis SEQ ID NO: 482 MC58 487 5.00E−75 conserved Neisseria NP_284304.1 nt. 2541-2978 of hypothetical protein meningitidis SEQ ID NO: 487 Z2491 488 2.00E−64 unknown Pasteurella NP_246617.1 nt. 2983-3540 of multocida SEQ ID NO: 488 488 8.00E−93 1-deoxy-D-xylulose Zymomonas AAD29659.1 nt. 1344-1880 of 5-phosphate mobilis SEQ ID NO: 488 reductoisomerase 491 5.00E−51 rubredoxin Clostridium AAB50346.1 compliment of oxidoreductase acetobutylicum nt. 1690-2439 of homolog SEQ ID NO: 491 492 1.00E−27 phosphotransferase Staphylococcus AAK83253.1 compliment of system enzyme aureus nt. 755-970 of IIA-like protein SEQ ID NO: 492 493 2.00E−84 unknown Actinobacillus AAC70895.1 nt. 3333-3935 of actinomycetemco- SEQ ID NO: 493 mitans 493 4.00E−49 unknown Helicobacter NP_223898.1 nt. 3345-4010 of pylori J99 SEQ ID NO: 493 493 9.00E−31 transcriptional Acinetobacter AAF20290.1 nt. 1885-2793 of factor MdcH calcoaceticus SEQ ID NO: 493 493 6.00E−30 HimA Pasteurella NP_245565.1 nt. 1129-1260 of multocida SEQ ID NO: 493 494 4.00E−85 putative prophage Yersinia pestis NP_404712.1 nt. 900-2099 of integrase SEQ ID NO: 494 494 4.00E−63 DNA Xylella fastidiosa NP_299063.1 compliment of methyltransferase 9a5c nt. 5544-6170 of SEQ ID NO: 494 494 6.00E−19 MODIFICATION Lactococcus lactis P34877 compliment of METHYLASE subsp. cremoris nt. 5019-6113 of SCRFIA SEQ ID NO: 494 497 0 transferrin-binding Haemophilus S70906 nt. 3251-4999 of protein 1 influenzae (strain SEQ ID NO: 497 PAK 12085) 50 5.00E−07 AcpP Pasteurella NP_246856.1 nt. 2-136 of multocida SEQ ID NO: 50 501 7.00E−50 conserved Vibrio cholerae NP_231403.1 compliment of hypothetical protein nt. 3649-4872 of SEQ ID NO: 501 501 0 type I restriction Vibrio cholerae NP_231400.1 compliment of enzyme HsdR, nt. 1551-3440 of putative SEQ ID NO: 501 501 4.00E−13 ATP-dependent Deinococcus NP_295921.1 compliment of DNA helicase radiodurans nt. 5317-5844 of RecG-related SEQ ID NO: 501 protein 501 5.00E−11 conserved Ureaplasma NP_077868.1 compliment of hypothetical urealyticum nt. 5098-5769 of SEQ ID NO: 501 504 2.00E−44 OUTER Haemophilus Q48218 compliment of MEMBRANE influenzae nt. 4681-5019 of PROTEIN P2 AG30010 SEQ ID NO: 504 PRECURSOR (OMP P2) 507 0 SpoT Pasteurella NP_245857.1 compliment of multocida nt. 3685-5316 of SEQ ID NO: 507 51 6.00E−87 glucosamine-- Vibrio cholerae NP_230141.1 nt. 30-470 of fructose-6- SEQ ID NO: 51 phosphate aminotransferase (isomerizing) 512 2.00E−28 dipeptide transport Yersinia pestis NP_407439.1 compliment of system permease nt. 1095-1580 of protein SEQ ID NO: 512 512 3.00E−82 SapC Pasteurella NP_245850.1 compliment of multocida nt. 730-1095 of SEQ ID NO: 512 514 9.00E−06 putative integral Campylobacter NP_281236.1 compliment of membrane protein jejuni nt. 577-684 of SEQ ID NO: 514 514 3.00E−11 orf, hypothetical Escherichia coli NP_286004.1 compliment of protein O157:H7 EDL933 nt. 449-568 of SEQ ID NO: 514 518 0 putative inner Neisseria NP_284893.1 nt. 92-1927 of membrane trans- meningitidis SEQ ID NO: 518 acylase protein Z2491 519 4.00E−30 hypothetical protein Mesorhizobium NP_108196.1 compliment of loti nt. 2221-3159 of SEQ ID NO: 519 519 2.00E−12 conserved Listeria innocua NP_471067.1 compliment of hypothetical protein nt. 3994-5241 of SEQ ID NO: 519 519 6.00E−20 hypothetical protein Mesorhizobium NP_108198.1 compliment of loti nt. 707-1552 of SEQ ID NO: 519 519 4.00E−26 putative Salmonella NP_455526.1 compliment of bacteriophage enterica subsp. nt. 3982-5163 of protein enterica serovar SEQ ID NO: 519 Typhi 52 3.00E−94 OUTER Haemophilus Q48218 nt. 45-788 of MEMBRANE influenzae SEQ ID NO: 52 PROTEIN P2 PRECURSOR (OMP P2) 520 0 excision nuclease Escherichia coli NP_418482.1 compliment of subunit A K12 nt. 6309-7745 of SEQ ID NO: 520 521 5.00E−08 zinc/manganese Rickettsia conorii NP_359651.1 nt. 2236-2652 of ABC transporter SEQ ID NO: 521 substrate binding protein 521  1.00E−140 unknown Pasteurella NP_245865.1| nt. 338-1390 of multocida SEQ ID NO: 521 521 1.00E−86 ORF_f432 Escherichia coli AAB40463.1 nt. 203-1390 of SEQ ID NO: 521 522 3.00E−22 unknown Pasteurella NP_246093.1 nt. 670-885 of multocida SEQ ID NO: 522 526 5.00E−33 exodeoxyribonuclease Yersinia pestis NP_404635.1 nt. 5582-6202 of V alpha chain SEQ ID NO: 526 526 1.00E−62 exodeoxyribonuclease Vibrio cholerae NP_231950.1 nt. 5675-6193 of V, 67 kDa SEQ ID NO: 526 subunit 527  1.00E−147 unknown Pasteurella NP_245980.1 nt. 4283-5203 of multocida SEQ ID NO: 527 527 0 Mfd Pasteurella NP_245978.1 nt. 7545-8759 of multocida SEQ ID NO: 527 527 0 transcription-repair Salmonella NP_455708.1 nt. 7611-8762 of coupling factor enterica subsp. SEQ ID NO: 527 (TrcF) enterica serovar Typhi 527 0 PROBABLE Ralstonia NP_519763.1 nt. 7611-8870 of TRANSCRIPTION- solanacearum SEQ ID NO: 527 REPAIR COUPLING FACTOR PROTEIN 528 1.00E−48 undecaprenyl Chlamydia NP_297109.1 nt. 2918-3712 of pyrophosphate muridarum SEQ ID NO: 528 synthetase 528 0 leucyl-tRNA Vibrio cholerae NP_230603.1 compliment of synthetase nt. 180-2822 of SEQ ID NO: 528 529  1.00E−104 DNA PRIMASE Legionella P71481 compliment of pneumophila nt. 3316-3960 of SEQ ID NO: 529 534 9.00E−29 putative integrase Salmonella NP_461690.1 nt. 4668-5009 of typhimurium LT2 SEQ ID NO: 534 534 6.00E−18 hypothetical protein Neisseria NP_283002.1 compliment of NMA0153 meningitidis nt. 5933-6337 of Z2491 SEQ ID NO: 534 534 2.00E−23 hypothetical protein Deinococcus NP_294868.1 nt. 6908-7654 of radiodurans SEQ ID NO: 534 534 1.00E−88 prophage CP4-57 Escherichia coli NP_417111.1 nt. 5057-5875 of integrase K12 SEQ ID NO: 534 535  1.00E−115 phosphate Buchnera sp. NP_240007.1 nt. 3385-4596 of acetyltransferase APS SEQ ID NO: 535 536 3.00E−35 cobalt membrane Actinobacillus AAD49727.1 compliment of transport protein pleuropneumoniae nt. 3531-4136 of CbiQ SEQ ID NO: 536 536 6.00E−37 unknown Pasteurella NP_245305.1 compliment of multocida nt. 6478-6921 of SEQ ID NO: 536 539 2.00E−26 Orf122 Chlorobium AAG12204.1 compliment of tepidum nt. 1778-2008 of SEQ ID NO: 539 540 1.00E−77 heat shock protein Neisseria NP_273864.1 compliment of HtpX meningitidis nt. 2567-3481 of MC58 SEQ ID NO: 540 541 0 IleS Pasteurella NP_246601.1 nt. 3167-4549 of multocida SEQ ID NO: 541 545 2.00E−09 DNA-BINDING Pectobacterium Q47588 nt. 3816-3977 of PROTEIN RDGB carotovorum SEQ ID NO: 545 545 2.00E−11 putative Sinorhizobium NP_437741.1 compliment of transposase meliloti nt. 2786-3019 of SEQ ID NO: 544 545 2.00E−07 Hypothetical 42.5 Escherichia coli BAA77933.1 compliment of kd protein in thrW- nt. 2614-2811 of argF intergenic SEQ ID NO: 545 region 545 4.00E−18 putative IS element Salmonella NP_454711.1 nt. 1955-2230 of transposase enterica subsp. SEQ ID NO: 545 enterica serovar Typhi 546 0 HEME/HEMOPEXIN- Haemophilus P45354 nt. 5551-7809 of BINDING influenzae SEQ ID NO: 546 PROTEIN 546 0 HEME/HEMOPEXIN Haemophilus P45356 nt. 3842-5536 of UTILIZATION influenzae SEQ ID NO: 546 PROTEIN B 546 0 HEME/HEMOPEXIN Haemophilus P45357 nt. 1638-3176 of UTILIZATION influenzae SEQ ID NO: 546 PROTEIN C 546 2.00E−12 HasR Pasteurella NP_246561.1 nt. 3149-3763 of multocida SEQ ID NO: 546 549 0 unknown Pasteurella NP_246821.1 nt. 2526-3512 of multocida SEQ ID NO: 549 549  1.00E−121 putative membrane Yersinia pestis NP_404859.1 nt. 605-1108 of protein SEQ ID NO: 549 549 0 unknown Pasteurella NP_246822.1 nt. 1122-1664 of multocida SEQ ID NO: 549 551  1.00E−157 type I restriction- Xylella fastidiosa NP_300016.1 compliment of modification 9a5c nt. 7396-8322 of system SEQ ID NO: 551 endonuclease 552  1.00E−100 valyl-tRNA Deinococcus NP_293872.1 compliment of synthetase radiodurans nt. 6691-8688 of SEQ ID NO: 552 552 0 VALYL-TRNA Haemophilus P36432 compliment of SYNTHETASE parainfluenzae nt. 5850-6647 of SEQ ID NO: 552 553 0 DNA-directed RNA Vibrio cholerae NP_229982.1 nt. 2668-6699 of polymerase, beta SEQ ID NO: 553 subunit 554 0 iron utilization Haemophilus T10887 nt. 991-2508 of protein B influenzae SEQ ID NO: 554 559  1.00E−100 PREPROTEIN Bacillus firmus P96313 nt. 3420-4472 of TRANSLOCASE SEQ ID NO: 559 SECA SUBUNIT 56 2.00E−23 RpL30 Pasteurella NP_246336.1 compliment of multocida nt. 656-832 of SEQ ID NO: 56 56 9.00E−13 RpS5 Pasteurella NP_246337.1 compliment of multocida nt. 843-1064 of SEQ ID NO: 56 560  1.00E−157 Na+/H+ antiporter Vibrio cholerae NP_231535.1 2 compliment of nt. 279-2989 of SEQ ID NO: 560 562 1.00E−72 putative biotin Yersinia pestis NP_404419.1 nt. 7862-8878 of sulfoxide reductase SEQ ID NO: 562 2 562  1.00E−125 restriction Neisseria CAA09003.1 nt. 2-985 of modification meningitidis SEQ ID NO: 562 system-R protein 563 0 IMMUNOGLOBULIN Haemophilus P45384 compliment of A1 PROTEASE influenzae HK715 nt. 4127-9508 of SEQ ID NO: 563 563 0 3- Schizosaccharo- O14289 nt. 1980-3983 of ISOPROPYLMALATE myces pombe SEQ ID NO: 563 DEHYDRATASE (IPMI) 564 2.00E−79 orf32 Haemophilus NP_536839.1 nt. 6241-6831 of phage HP2 SEQ ID NO: 564 564 7.00E−33 probable variable Salmonella NP_457882.1 nt. 3707-4177 of tail fibre protein enterica subsp. SEQ ID NO: 564 enterica serovar Typhi 564 2.00E−14 M protein Enterobacteria NP_052264.1 nt. 1905-2213 of phage 186 SEQ ID NO: 564 564 4.00E−44 similar to tail fiber Salmonella NP_461635.1 nt. 3171-3692 of protein (gpH) in typhimurium LT2, SEQ ID NO: 564 phage P2 Fels-2 prophage 564 2.00E−85 gpJ Enterobacteria NP_046773.1 nt. 2267-3166 of phage P2 SEQ ID NO: 564 564 1.00E−24 hypothetical protein Neisseria NP_284534.1 nt. 6852-7334 of meningitidis SEQ ID NO: 564 Z2491 564 4.00E−26 gpv Enterobacteria NP_046771.1 nt. 1337-1912 of phage P2 SEQ ID NO: 564 564 2.00E−47 similar to Escherichia coli BAA16182.1 nt. 11383-11961 of [SwissProt P44255 SEQ ID NO: 564 564 2.00E−51 hypothetical protein Neisseria NP_284066.1 nt. 10452-11180 NMA1315 meningitidis of SEQ ID NO: 564 Z2491 564 0 orf31 Haemophilus NP_043502.1 nt. 4160-6226 of phage HP1 SEQ ID NO: 564 564 2.00E−09 rep Haemophilus NP_536816.1 compliment of phage HP2 nt. 9986-10234 of SEQ ID NO: 564 565 2.00E−57 resolvase/ Haemophilus AAL47097.1 nt. 11885-12445 of integrase-like influenzae biotype SEQ ID NO: 565 protein aegyptius 565 1.00E−93 integrase Actinobacillus AAC70901.1 compliment of actinomycetemco- nt. 4118-4900 of mitans SEQ ID NO: 565 565 6.00E−35 probable phage Salmonella NP_458745.1 compliment of integrase enterica subsp. nt. 4148-4990 of enterica serovar SEQ ID NO: 565 Typhi 565  1.00E−107 hypothetical protein Xylella fastidiosa NP_299042.1 compliment of 9a5c nt. 5066-6817 of SEQ ID NO: 565 566  1.00E−126 hypothetical protein Haemophilus S15287 compliment of (galE 5′ region) - influenzae nt. 10726-11607 of SEQ ID NO: 566 567 0 unknown Pasteurella NP_246387.1 nt. 5343-7688 of multocida SEQ ID NO: 567 568  1.00E−151 multidrug Escherichia coli NP_311575.1 nt. 6-1403 of resistance O157:H7 SEQ ID NO: 568 membrane translocase 568  1.00E−141 YhbX/YhjW/YijP/Yj Neisseria |NP_275002.1 compliment of dB family protein meningitidis nt. 11213-12634 of MC58 SEQ ID NO: 568 570  1.00E−180 hypothetical protein Haemophilus S71024 compliment of 3 (ksgA-lic2B influenzae (strain nt. 12845-13720 of intergenic region) RM7004) SEQ ID NO: 570 571 0 glycerophospho- Haemophilus A43576 nt. 1656-2693 of diester influenzae (isolate SEQ ID NO: 571 phosphodiesterase 772) 571  1.00E−137 outer membrane Haemophilus A43604 nt. 6145-6909 of protein P4 influenzae SEQ ID NO: 571 precursor - Haemophilus influenzae 571 2.00E−72 CG8298 gene Drosophila AAF58597.1 nt. 3813-5339 of product [alt 1] melanogaster SEQ ID NO: 571 572 1.00E−40 hypothetical protein Chlamydia G81737 nt. 3734-4099 of TC0130 muridarum (strain SEQ ID NO: 572 Nigg) 572 5.00E−10 hypothetical protein Pyrococcus NP_142215.1 nt. 4472-4888 of horikoshii SEQ ID NO: 572 572 3.00E−11 109aa long Sulfolobus NP_377117.1 nt. 7303-7470 of hypothetical protein tokodaii SEQ ID NO: 572 572 8.00E−43 hypothetical protein Chlamydophila NP_445524.1 nt. 4289-4618 of pneumoniae SEQ ID NO: 572 AR39 572 9.00E−08 CDH1-D Gallus gallus AAL31950.1 nt. 7183-7521 of SEQ ID NO: 572 575  1.00E−173 topoisomerase B Salmonella NP_458624.1 nt. 18980-20923 of enterica subsp. SEQ ID NO: 575 enterica serovar Typhi 575  1.00E−100 DNA helicase Salmonella NP_458617.1 nt. 10399-11706 of enterica subsp. SEQ ID NO: 575 enterica serovar Typhi 65 2.00E−53 Sufl Pasteurella NP_245041.1 nt. 3-821 of multocida SEQ ID NO: 65 67 4.00E−39 putative MFS Salmonella NP_462786.1 compliment of family tranport typhimurium LT2 nt. 125-1033 of protein (1st mdule) SEQ ID NO: 67 7 4.00E−29 putative membrane Salmonella NP_458664.1 compliment of protein enterica subsp. nt. 2-559 of enterica serovar SEQ ID NO: 7 Typhi 72 2.00E−51 serine transporter Vibrio cholerae NP_230946.1 nt. 18-803 of SEQ ID NO: 72 74 3.00E−90 hypothetical 21.8K Haemophilus JH0436 compliment of protein (in locus influenzae nt. 248-766 of involved in SEQ ID NO: 74 transformation) - 77 2.00E−18 RecX protein Legionella CAC33485.1 nt. 480-920 of pneumophila SEQ ID NO: 77 82 4.00E−95 unknown Pasteurella NP_246414.1 nt. 128-955 of multocida SEQ ID NO: 82 83 2.00E−66 unknown Pasteurella NP_246777.1 nt. 5-556 of multocida SEQ ID NO: 83 83 6.00E−33 CTP SYNTHASE Helicobacter NP_223042.1 compliment of pylori J99 nt. 1027-1338 of SEQ ID NO: 83. 83 4.00E−34 CTP synthase Campylobacter NP_281249.1 compliment of jejuni nt. 1024-1275 of SEQ ID NO: 83 84 1.00E−16 REPRESSOR Bacteriophage P14819 nt. 823-1233 of PROTEIN CI phi-80 SEQ ID NO: 84 84 2.00E−05 orf, hypothetical Escherichia coli NP_415875.1 compliment of protein K12 nt. 533-700 of SEQ ID NO: 84 84 4.00E−11 orf33 bacteriophage phi NP_490633.1 compliment of CTX nt. 32-466 of SEQ ID NO: 84 85 3.00E−42 SpoT Pasteurella NP_245857.1 nt. 899-1261 of multocida SEQ ID NO: 85 90  1.00E−103 putative methylase Bacteriophage NP_108695.1 compliment of Tuc2009 nt. 478-1206 of SEQ ID NO:90 90 4.00E−11 probable adenine Thermoplasma NP_394624.1 compliment of specific DNA acidophilum nt. 397-1140 of methyltransferase SEQ ID NO: 90

TABLE 3B Full Length Nucleotide Amino Acid Homology to Genbank Hit Identity Sequence Sequence Location in Contig Protein CpdB SEQ ID NO: SEQ ID NO: nt. 38041-36068 of NP_246953.1 686 687 SEQ ID NO: 681 (contig 14) putative membrane SEQ ID NO: SEQ ID NO: nt. 906601-908094 NP_458664.1 protein 688 689 of SEQ ID NO: 685 (contig 18) GTP-binding SEQ ID NO: SEQ ID NO: nt. 42557-40995 of NP_240245.1 protein TypA/BipA 690 691 SEQ ID NO: 683 (contig 16) outer membrane SEQ ID NO: SEQ ID NO: nt. 7000420-704187 T30852 protein A 692 693 of SEQ ID NO: 685 (contig 18) vacB protein SEQ ID NO: SEQ ID NO: nt. 39184-36836 of NP_240369.1 694 695 SEQ ID NO: 683 (contig 16) putative ABC SEQ ID NO: SEQ ID NO: nt. 59155-58370 of NP_282774.1 transport system 696 697 SEQ ID NO: 685 permease protein [ (contig 18) putative exported SEQ ID NO: SEQ ID NO: nt. 901142-902542 NP_458655.1 protein 698 699 of SEQ ID NO: 685 (contig 18) ImpA SEQ ID NO: SEQ ID NO: nt. 348187-347747 NP_245829.1 700 701 of SEQ ID NO: 685 (contig 18) TsaA SEQ ID NO: SEQ ID NO: nt. 74941-75548 of NP_245732.1 702 703 SEQ ID NO: 684 (contig 17) PROBABLE SEQ ID NO: SEQ ID NO: nt. 74436-75176 of NP_522358.1 TRANSPORT 704 705 SEQ ID NO: 685 TRANSMEMBRANE (contig 18) PROTEIN SEQ ID NO: SEQ ID NO: nt. 75160-75660 of 706 707 SEQ ID NO: 685 (contig 18) possible exported SEQ ID NO: SEQ ID NO: nt. 899618-900262 NP_458653.1 protein 708 709 of SEQ ID NO: 685 (contig 18) LICA PROTEIN SEQ ID NO: SEQ ID NO: nt. 356917-355958 P14181 710 711 of SEQ ID NO: 685 (contig 18) HEME-BINDING SEQ ID NO: SEQ ID NO: NT. 26114-27739 P33950 PROTEIN A 712 713 of SEQ ID NO: 683 (contig 16) similar to SEQ ID NO: SEQ ID NO: nt. 311610-312683 XP_068727.1 BASEMENT 714 715 of SEQ ID NO: 685 MEMBRANE- (contig 18) SPECIFIC HEPARAN SULFATE PROTEOGLYCAN CORE PROTEIN PRECURSOR (HSPG) CzcD SEQ ID NO: SEQ ID NO: nt. 34865-35542 of NP_246276.1 716 717 SEQ ID NO: 681 (contig 14) conserved SEQ ID NO: SEQ ID NO: nt. 194993-193977 NP_274972.1 hypothetical protein 718 719 of SEQ ID NO: 685 (contig 18) secretion protein SEQ ID NO: SEQ ID NO: nt. 203707-201857 NP_252510.1 SecD 720 721 of SEQ ID NO: 683 (contig 17) ABC transporter SEQ ID NO: SEQ ID NO: nt. 3943-5859 of AAF31030.1 protein 1 722 723 SEQ ID NO: 681 (contig 14) conserved SEQ ID NO: SEQ ID NO: nt. 331090-331749 NP_273467.1 hypothetical protein 724 725 of SEQ ID NO: 685 (contig 18) SEQ ID NO: SEQ ID NO: nt. 331938-332492 726 727 of SEQ ID NO: 685 (contig 18) SEQ ID NO: SEQ ID NO: nt. 332681-33232 728 729 of SEQ ID NO: 685 (contig 18) INVASIN SEQ ID NO: SEQ ID NO: nt. 416757-417020 P31489 PRECURSOR 730 731 of SEQ ID NO: 685 (OUTER (contig 18) MEMBRANE ADHESIN) HEME/HEMOPEXIN- SEQ ID NO: SEQ ID NO: nt. 229430-232195 P45355 BINDING 732 733 of SEQ ID NO: 384 PROTEIN (contig 17) OPACITY SEQ ID NO: SEQ ID NO: nt. 375592-375879 Q05033 PROTEIN OPA66 734 735 of SEQ ID NO: 384 PRECURSOR (contig 17) Hemoglobin- SEQ ID NO: SEQ ID NO: nt. 45709-42566 of Q48153 haptoglobin binding 736 737 SEQ ID NO: 681 protein A (contig 14) transport protein SEQ ID NO: SEQ ID NO: nt. 134452-135222 NP_253757.1 TatC 738 739 of SEQ ID NO: 384 (contig 17) LIPOPROTEIN SEQ ID NO: SEQ ID NO: nt. 18895-20112 of P40827 NLPD 740 741 SEQ ID NO: 682 (contig 15) Hemoglobin and SEQ ID NO: SEQ ID NO: nt. 34181-31041 of Q9X442 hemoglobin- 742 743 SEQ ID NO: 682 haptoglobin binding (contig 15) protein C precursor HimA SEQ ID NO: SEQ ID NO: nt. 382795-383085 NP_245565.1 744 745 of SEQ ID NO: 685 (contig 18) transferrin-binding SEQ ID NO: SEQ ID NO: nt. 178537-175799 S70906 protein 1 746 747 of SEQ ID NO: 683 (contig 16) SapC SEQ ID NO: SEQ ID NO: nt. 197754-196867 NP_245850.1 748 749 of SEQ ID NO: 685 (contig 18) heat shock protein SEQ ID NO: SEQ ID NO: nt. 40414-41265 of NP_273864.1 HtpX 750 751 SEQ ID NO: 682 (contig 15) HEME/HEMOPEXIN- SEQ ID NO: SEQ ID NO: nt. 229430-232195 P45354 BINDING 752 753 of SEQ ID NO: 684 PROTEIN (contig 17) HEME/HEMOPEXIN SEQ ID NO: SEQ ID NO: nt. 227721-229418 P45356 UTILIZATION 754 755 of SEQ ID NO: 684 PROTEIN B (contig 17) HEME/HEMOPEXIN SEQ ID NO: SEQ ID NO: nt. 225516-227645 P45357 UTILIZATION 756 757 of SEQ ID NO: 684 NP_246561.1 PROTEIN C (contig 17) iron utilization SEQ ID NO: SEQ ID NO: nt. 32076-33611 of T10887 protein B 758 759 SEQ ID NO: 684 (contig 17) PREPROTEIN SEQ ID NO: SEQ ID NO: nt. 82314-84785 of P96313 TRANSLOCASE 760 761 SEQ ID NO: 683 SECA SUBUNIT (contig 16) IMMUNOGLOBULIN SEQ ID NO: SEQ ID NO: nt. 171647-166263 P45384 A1 PROTEASE 762 763 of SEQ ID NO: 683 (contig 16) multidrug SEQ ID NO: SEQ ID NO: nt. 74524-72992 of NP_311575.1 resistance 764 765 SEQ ID NO: 683 membrane (contig 16) translocase YhbX/YhjW/YijP/YjdB SEQ ID NO: SEQ ID NO: nt. 61734-63200 of NP_275002.1 family protein 766 767 SEQ ID NO: 683 (contig 16) putative membrane SEQ ID NO: SEQ ID NO: nt. 906601-908094 NP_458664.1 protein 768 769 of SEQ ID NO: 685 (contig 18) putative membrane SEQ ID NO: SEQ ID NO: nt. 16185-17942 of NP_404859.1 protein 770 771 SEQ ID NO: 683 (contig)

Example 3 Construction of the NTHi Promoter Trap Library

To identify potential virulence determinants of NTHi, bacterial gene expression was monitored by differential fluorescence induction (DFI) during early disease progression in one specific anatomical niche of a chinchilla model of NTHi-induced otitis media (OM). Genomic DNA fragments from NTHi strain 86-028NP were cloned upstream of the promoterless gfpmut3 gene using a promoter trap library. Plasmid pGZRS39A, a derivative of pGZRS-1 isolated from Actinobacillus pleuropneumoniae, is an A. pleuropneumoniae-Escherichia coli shuttle vector. This plasmid contains the origin of replication from A. pleuropneumoniae, the lacZα gene from pUC19 and the kanamycin resistance gene from Tn903. (West et al., Genes, 160: 81-86, 1995).

The promoter trap vector was constructed by cloning the GTP mutant gfpmut3 gene, as a BamHI to EcoRI fragment into pGZRS-39A to form pRSM2167. This mutant GTP gene contains two amino acid changes, S65G and S72A, that enhance fluorescence emission when excited at 488 nm. This mutant also has high solubility and fast kinetics of chromophore formation (Cormack et al., Gene, 173: 33-38, 1996). This plasmid was transformed by electroporation into NTHi strain 86-028NP, generating the parent-plasmid strain 86-028NP/pRSM2169.

Random genomic DNA fragments (described in Example 1) were prepared for ligation into the promoter probe vector. Genomic DNA was isolated from strain 86-028NP using the Puregene DNA isolation kit (Gentra Systems, Minneapolis, Minn.) according to the manufacturer's protocol. Due to restriction barriers, it was necessary to isolate the plasmid DNA and use this for the library generation. The isolated DNA was partially digested with Sau3AI (NEB, Beverly, Mass.; 0.25 units/μg DNA) for 1 hour at 37° C., separated by gel electrophoresis and DNA fragments 0.5-1.5 kb in size were recovered using the Qiagen gel extraction kit. For vector preparation, pRSM2167 was isolated from an overnight culture using the Wizard Plus Maxiprep DNA purification system (Promega, Madison Wis.) according to the manufacturer's protocol.

Plasmid DNA was linearized by BamHI digestion and 5′ phosphate groups removed by treatment with calf intestinal alkaline phosphatase (CLAP; GibcoBRL Life Technologies). Genomic DNA fragments were ligated with the linearized, phosphatase-treated vector and electroporated into competent NTHi strain 86-028NP prepared for electroporation according to a modified protocol (Mitchell et al., Nucleic Acids Res., 19: 3625-3628, 1991). When plasmid DNA was electroporated back into NTHi strain 86-028NP, transformation efficiency was improved by one-thousand fold. Briefly, cells were grown to an OD₆₀₀=0.3 in sBHI (brain heart infusion) broth at 37° C., 220 rpm. Cells were chilled on ice for 30 minutes and subsequently washed with an equal volume of 0.5×SG (1×SG: 15% glycerol, 272 mM sucrose) at 4° C. Washes were repeated a total of three times. Subsequently, the cells were diluted in 1×SG to a 100× concentrated volume. The cells were electroporated using the BioRad Gene Pulser II set at 200 ohms, 2.5 kV and 25 μF and then diluted in 1 ml prewarmed sBHI, incubated for 2 hours at 37° C., 5% CO₂ and plated on chocolate agar for overnight growth of transformants.

Transformants were selected and frozen in pools of 1000 clones in skim milk containing 20% glycerol (vol/vol). A 68,000 member gfp promoter probe library was generated. Using the probability calculation of Clarke and Carbon (Cell, 9: 91-99, 1976), to achieve a 99% probability of having a given DNA sequence represented in a library of 300 bp fragments of strain 86-028NP DNA (1.8×10⁶ bp/genome), a library of 27,629 clones was needed. Therefore the present library represents 2.5 fold coverage of the 86-028NP genome.

In order to assess the quality of the library, fifty clones were selected at random, grown overnight on chocolate agar and the plasmids were isolated and insert DNA sequenced. A majority (64%) of the selected clones had insert sizes ranging between 200 and 500 bp while 32% exceeded 500 bp. The majority of inserts showed homology to unique H. influenzae strain Rd open reading frames (ORFs), and 15 clones had sequence unique to strain 86-028NP DNA. Of those clones with homology to strain Rd, 60% were in the correct orientation, 36% of which contained sequence upstream an ORF. Although a majority of clones had an insert size less than 500 bp, no correlation was found between small insert size and increased GFP expression. In fact four clones exhibited slight to moderate fluorescence in vitro, 3 of which had insert sizes between 200-500 base pairs and one had an insert that was greater than 700 base pairs.

A fraction of the library (approximately 1000 clones) was grown on chocolate agar, harvested in PBS and analyzed by flow cytometry for GFP fluorescence. Compared to strain 86-028NP/pRSM2169 that contains the promoter trap vector without insert DNA, the pool of library clones displays an increased fluorescence intensity. Thus, the library contains clones with promoters at varying levels of activity.

Example 4 Analysis of 86-028NP Derivatives Expressing GFP

In order to establish the FACS parameters necessary to identify and sort gfp-expressing bacteria, a panel of isolates demonstrating varying levels of gfp expression was utilized. Background fluorescence was assessed using strain 86-028NP/pRSM2169 (negative control), therefore any observed fluorescence would be due to the lacZ promoter driving gfp expression. However, this strain does not produce detectable levels of GFP and in fact, does not demonstrate increased fluorescence when compared to the parent strain 86-028NP. A high-level gfp-expressing isolate was generated by cloning a 500 bp fragment containing the strong promoter for outer membrane protein P2 expression into SalI-BamHI digested pRSM2167. This plasmid was transformed into 86-028NP by electroporation, generating the high-level gfp expressing strain 86-028NP/pRSM2211 (highly fluorescent control). This strain demonstrated an approximate 100 fold increase in GFP fluorescence compared to strain 86-028NP/pRSM2169. An intermediate fluorescent derivative clone, 86-028NP/pKMM4B5 (intermediate fluorescent control), was isolated by FACS analysis and used both in preliminary experiments and as a control for cell sorting. The DNA fragment containing a promoter driving gfp expression in vitro is unique to strain 86-028NP, having no known homology to DNA of other organisms. This clone exhibits an approximate 10 fold increase in fluorescence compared to strain 86-028NP/pRSM2169.

The control strains were resuspended from growth on chocolate agar and labeled with cross-reactive Phycoprobe R-PE anti-human IgG (H+ L) antibody (10 μg/ml in 100 μl PBS; Biomeda Corp) for 30 minutes at 4° C. Following three successive washes to remove unbound antibody, bacteria were resuspended in 300 μl DPBS for FACS analysis. These control preparations were used to set the appropriate size and fluorescence gates using a Coulter Epics Elite flow cytometer (Coulter Corp.) equipped with an argon laser emitting at 488 nm. Bacteria were gated for size based on log forward angle and side scatter detection and for sorting by FITC/PE labeling of bacteria. Sorted cells were collected into cold sBHI and plated on chocolate agar. After overnight growth, cells were collected for a secondary round of infection or were individually selected and grown overnight, screened by individual clone for fluorescence when grown in vitro, and frozen in skim milk containing 20% (vol/vol) glycerol prior to plasmid isolation and sequencing of insert DNA. Sorting efficiency of control strains was confirmed using a Coulter EPICS flow cytometer (Coulter Corp.).

Many plasmids were segregated rapidly in vitro in the absence of antibiotic selection. Thus, in order to assess whether the promoter trap vector used here was prone to this event, a single colony of strain 86-028NP/pRSM2211 (highly fluorescent control) was isolated on chocolate agar and passaged 20 times in the absence of antibiotic selection. No significant decrease in fluorescence intensity was observed when compared to bacteria grown in the presence of antibiotic. In addition, the plasmid is maintained in the absence of antibiotic selection in vivo. Similar bacterial counts were observed when bacteria-containing middle ear fluids collected from a chinchilla were plated on chocolate agar with or without kanamycin. These data demonstrate that the promoter trap vector was stably maintained in the absence of antibiotic selection.

In addition to problems with plasmid stability, early studies on the use of GFP as a reporter to study host-pathogen interactions demonstrated that GFP could be continuously synthesized as a cytoplasmic protein with low toxicity, having minimal effects on the bacterial cell-surface dynamics (Chalfie et al., Science, 263: 802-805, 1994). The construction of a high level gfp-expressing derivative allowed the assessment of the GFP toxicity on NTHi. Growth curves of both the wild-type strain (86-028NP) and the high GFP producing strain 86-028NP/pRSM2211 were compared when grown under similar conditions. The growth rates were similar, indicating that GFP expression was not toxic to the cells.

The 86-028NP gfp-expressing derivatives were used to define the parameters for efficient cell sorting. Strain 86-028NP/pRSM2169 was mixed with the intermediate gfp-expressing derivative, strain 86-028NP/pKMM4B5, at a 100:1 ratio, simulating the in vivo environment that is expected to contain a small percentage of gfp-expressing clones relative to the total bacterial population. This mixture was subjected to FACS analysis, collecting the 1.8% most fluorescent population and the 52% least fluorescent population. Flow cytometric analysis of the sorted populations revealed an enrichment of strain 86-028NP/pKMM4B5 to 65% of the bacterial population, a phenomenon that was not observed when sorting on the negative population. Subsequent rounds of sorting would be expected to further enrich for this intermediate fluorescent population. The inability to decrease the amount of fluorescent bacteria in the negative sort was attributed to the size of the gate set for negative sorting. GFP-negative cells were enriched by gating on the 10% least fluorescent population.

Example 5 Direct Labeling of Bacteria from Middle Ear Fluids

A similar strategy (as described in Example 5) was applied to sort fluorescent clones from effusions obtained from the chinchilla middle ear during AOM. Our ability to use differential fluorescence induction (DFI) in vivo was dependent upon our ability to sort gfp-expressing bacteria from non-fluorescent bacteria, fluorescent and non-fluorescent cellular debris, and eukaryotic cells.

Healthy adult chinchillas (Chinchilla lanigera) with no evidence of middle ear infection by either otoscopy or tympanometry were used to screen the library for promoter activity in vivo. Two pools of the NTHi/pRSM2169 library (1000 clones each) were grown overnight on chocolate agar containing kanamycin. The library was combined and diluted in cold 10 mM sterile PBS to 3.3×10⁶ CFU/ml and 300 μl (1.0×10⁶ CFU; 500 CFU/clone) was used to inoculate the left and the right chinchilla transbullar cavity (2000 clones/ear). OM development was monitored by video otoscopy and tympanometry at 24 and 48 hours. The bacteria multiplied in the middle ear cavity, reaching a concentration 500 times the inoculum dose by 48 hours as expected (Bakaletz et al., Infect. Immunity 67: 2746-62, 1999). This bacterial adaptation to the host environment results in an inflammatory response, indicated by erythema, vessel dilation and bulging of the tympanic membrane, infiltration of polymorphonuclear cells (PMN's), and accumulation of fluid in the middle ear cavity as observed by otoscopy and microscopic examination of recovered effusions. Twenty-four and 48 hours later, middle ear fluids were retrieved by epitympanic tap, and prepared for FACS.

It is important to note that this analysis was limited to those bacteria recoverable in the middle ear fluid. In some cases it was necessary to lavage the middle ear cavity to collect the bacteria for FACS analysis. Thus, this analysis includes genes up-regulated when NTHi are loosely adherent to mucosae. NTHi has been observed to form a biofilm in the middle ear cavity in a chinchilla model of OM (Erhlich et al., JAMA, 287: 1710-5, 2002). Since the protocols described herein select for clones recovered from the planktonic population, it is not expected to recover those clones in which genes are up-regulated when the bacteria are associated with mucosal biofilms. Homogenization of middle ear mucosae and subsequent bacterial cell isolation however, would enable us to recover these clones. It is also possible that some GFP-expressing clones were recovered in the effusion, yet were adherent to eukaryotic cells present in the effusion as exfoliated cells, or in aggregates. These bacteria are difficult to recover from the effusion without compromising the sorting efficiency. Therefore the middle ear fluids were treated with a mucolytic agent, then centrifuged to remove large aggregates and eukaryotic cells and prior to labeling.

Chinchilla middle ear fluids were diluted, if necessary, to 250 μl with sterile saline. An equal volume of N-acetyl-L-cysteine (0.5%; w/v) in DPBS (pH 7.4) was added for 5 minutes at room temperature as a mucolytic agent (Miyamoto and Bakaletz, Microb. Pathog., 21: 343-356 1996). Fluids were centrifuged (300×g, 5 min) to remove cellular debris, red blood cells and inflammatory cells, and supernatants containing bacteria were transferred to a fresh tube. Bacteria were incubated with chinchilla antiserum (1:50 dilution) directed against a whole OMP preparation, derived from NTHi strain 86-028NP, for 45 minutes at 4° C., pelleted by centrifugation (2000×g, 5 min) and washed twice with cold DPBS containing 0.05% bovine serum albumin. Bacteria were subsequently labeled with cross-reactive phycoprobe R-PE anti-human IgG (H+ L) antibody (10 μg/ml in 100 μl PBS; Biomeda Corp) for 30 minutes at 4° C. Following three successive washes to remove unbound antibody, cells were resuspended in 300 μl DPBS for FACS analysis.

Example 6 Identification of Promoters Induced In Vivo in Acute Otitis Media

H. influenzae 86-028NP transformed with the promoter trap library was grown overnight on chocolate agar. To select against those clones containing promoters that expressed gfp in vitro, the library was subjected to one round of FACS analysis (as described in Example 6), collecting only those clones expressing low-level amounts of GFP. These clones were pooled and used to inoculate the chinchilla middle ear transbullarly. Following 24 and 48 hours of infection, bacteria-containing effusions were removed by epitympanic tap. Bacteria were indirectly labeled with R-PE-labeled antibody and subjected to FACS analysis by gating on fluorescently tagged bacteria but sorting for those that were also expressing. These clones were used to reinfect animals for further enrichment. Following the final round of sorting, single colony isolates were screened in vitro for lack of fluorescence.

Those clones isolated by FACS analysis (positive for GFP fluorescence in vivo), which did not emit fluorescence in vitro were prepared for plasmid isolation and identification of insert DNA sequence. These clones were grown overnight on chocolate agar plates containing kanamycin and prepared for plasmid isolation using the Qiaprep Miniprep Kit (Qiagen) according to the manufacturer's protocol. Plasmid insert DNA was sequenced using the primer 5′-TGCCCATTAACATCACCATCTA-3′ (SEQ ID NO: 588) that is complementary to the gfpmut3 gene and downstream of the insert DNA. Sequencing reactions were performed using the ABI prism BigDye® terminator cycle sequencing ready reaction kit (Applied Biosystems) according to manufacturer's protocol using a GeneAmp PCR System 9700 (Applied Biosystems). The sequences were then purified by passage through sephadex G-50 in a 96-well multiscreen HV plate (Millipore) and subsequently analyzed on an ABI Prism 3100 DNA analyzer (Applied Biosystems).

Insert sequences were compared to the complete annotated sequence of H. influenzae strain Rd. Those inserts with no nucleotide homology to strain Rd were subsequently analyzed using the BLASTN and BLASTX algorithms. Further sequence analysis was performed with DNASTAR (Madison, Wis.). Inserts in the correct orientation and containing sequence 5′ to a predicted ORF contained a putative promoter that was preferentially active when the NTHi bacteria were in the chinchilla middle ear.

Fifty-two clones with putative promoters that were regulated in vivo were isolated. Of the 44 candidate clones containing sequence similar to that identified in H. influenzae strain Rd, quantitative comparison of gene expression in vitro and in vivo confirmed up-regulated gene expression for twenty-six genes (60%) when NTHi respond to environmental cues present in the chinchilla middle ear and these genes are summarized in Table 4A below. The in vivo-regulated promoters driving expression of genes are predicted to be involved in membrane transport, environmental informational processing, cellular metabolism, gene regulation, as well as hypothetical proteins with unknown function.

In order to confirm the induction of putative promoter candidates in vivo, the relative amount of messenger RNA expression was compared when NTHi strain 86-028NP was grown in vitro to mid-log phase or in vivo for 48 hours. The RNA was isolated using TRIzol LS reagent (Gibco Life Technologies) according to the manufacturer's protocol. DNA was removed from the RNA preparation using DNA-free kit (Ambion) according to the manufacturer's protocol. DNase I treated RNA samples were purified by passage through a Qiagen RNeasy column. RNA purity and integrity was assessed by 260/280 nm spectrophotometer readings and on the Agilent 2100 Bioanalyzer (Agilent Technologies), respectively.

In order to independently confirm the FACS data, we determined the relative expression of candidate genes by quantitative RT-PCR. The parent strain 86-028NP, was used for these studies. Real-time quantitative RT-PCR using the one-step QuantiTect SYBR Green RT-PCR kit (Qiagen) assessed transcription levels according to the manufacture's instructions. Briefly, using primers generated to an open reading frame downstream of the putative in vivo-induced promoters identified by FACS analysis, gene-specific mRNA was reverse transcribed and amplified by RT-PCR on the ABI Prism 7700 sequence detection system (Applied Biosystems). The amount of product was calculated using a standard curve generated to known amounts of bacterial genomic DNA (10²-10⁷ genomic copies DNA) by amplifying a fragment of the gyrase (gyr) gene. Controls were analyzed in parallel to verify the absence of DNA in the RNA preparation (—RT control) as well as the absence of primer dimers in control samples lacking template RNA. In addition, RT-PCR products were analyzed by gel electrophoresis and, in all cases, a single product was observed at the appropriate base pair size. Amounts of bacterial RNA between samples were normalized relative to gyr expression, shown to be constitutively expressed under various growth conditions that we tested in vitro. Known amounts of bacterial genomic DNA (10²-10⁷ genomic copies DNA) were used to generate a standard curve for RT-PCR quantitation by amplifying a fragment of the gyrase (gyr) gene. Gyrase is constitutively expressed in vitro under various growth conditions and was therefore used to normalize total bacterial RNA levels between samples. Relative gene expression in vivo was compared to that of gene expression in vitro and data expressed as fold-increase are summarized in Table 4A.

The 8-fold sequencing of the NTHi genome identified the full length open reading frames for the majority of genes listed in Table 4A. Table 4B provides the full length nucleotide sequence within the NTHi genome and the corresponding amino acid sequence. The fold induction of the gene due to environmental cues present in the chinichilla middle ear and the product or function of the gene are repeated in Table 4B for convenience.

TABLE 4A SEQ Gene or ID GenBank Fold Category ORF NO: Protein ID Induction Product or Function Amino acid hisB 589 NP_438632 2.9 Histidine biosynthesis metabolism bifunctional protein Lipoprotein lppB 590 NP_438862.1 2.6 Lipoprotein B homologue Membrane transport sapA 591 NP_439780.1 2.8 Peptide ABC transporter; periplasmic SapA precursor lolA 592 NP_439736.1 2.4 Outer membrane lipoproteins carrier protein precursor rbsC 593 NP_438661.1 5.1 Ribose transport system permease protein Purine synthesis purE 594 NP_439757.1 51.7 Phosphoribosylaminoimidazole carboxylase catalytic subunit; PurE Biosynthetic and ribB 595 NP_438923.1 8.3 3,4-dihydroxy-2-butanone 4- metabolic functions phosphate synthase; riboflavin biosynthesis arcB 596 NP_438753.1 10 Ornithine carbamolytransferase; arginine degradation uxuA 597 NP_438228.1 3.1 Mannonate dehydratase; production of glyceraldehyde 3-phosphate dsbB 598 NP_438589.1 2.6 Disulfide oxidoreductase; disulfide bond formation protein B ureH 599 NP_438693.1 3.9 Urease accessory protein licC 600 NP_439688.1 2.3 Phosphocholine (ChoP) cytidylyltransferase HI1647 601 NP_439789.1 2.0 Putative pyridoxin biosynthesis protein; singlet oxygen resistance protein DNA replication, ispZ 602 P43810 2.5 Probable intracellular repair septation protein radC 603 NP_439113.1 2.1 DNA repair protein mukF 604 P45185 2.0 MukF protein homologue; remodeling of nucleiod structure Gene regulation glpR 605 NP_438777.1, 2.8 Glycerol-3-phosphate regulon NP_439170.1 repressor ihfB 606 P43724 2.5 Integration host factor beta subunit argR 607 NP_439365.1 2.7 Arginine repressor cspD 608 NP_439584.1 2.1 Cold shock like protein; stress response protein Hypothetical or HI0094 609 NP_438267.1 8.3 Hypothetical protein unknown HI1163 610 NP_439321.1 2.3 Conserved hypothetical proteins protein; putative oxidase HI1063 611 NP_439221.1 2.7 Hypothetical protein HI0665 612 NP_438824.1 2.8 Hypothetical protein HI1292 613 NP_439444.1 2.6 Hypothetical protein HI1064 614 NP_439222.1 2.6 Hypothetical protein

TABLE 4B Full Gene Length or Nucleotide Amino Acid Fold Product or Category ORF Sequence Sequence Location in Contig Induction Function Amino hisB SEQ ID NO: SEQ ID NO: nt. 68378-67290 2.9 Histidine acid 615 616 of SEQ ID NO: biosynthesis metabolism 680 (contig 13) bifunctional protein Membrane sapA SEQ ID NO: SEQ ID NO: nt. 200403-198709 2.8 Peptide ABC transport 617 618 of SEQ transporter; ID NO: 685 periplasmic SapA (contig 18) precursor rbsC SEQ ID NO: SEQ ID NO: nt. 42773-41802 5.1 Ribose transport 619 620 of SEQ ID NO: system permease 680 (contig 13) protein Purine purE SEQ ID NO: SEQ ID NO: nt. 219625-219131 51.7 Phosphoribosylaminoimidazole synthesis 621 622 of SEQ carboxylase catalytic ID NO: 685 subunit; PurE (contig 18) Biosynthetic ribB SEQ ID NO: SEQ ID NO: nt. 131537-132184 8.3 3,4-dihydroxy-2- and 623 624 of SEQ butanone 4- metabolic ID NO: 682 phosphate synthase; functions (contig 15) riboflavin biosynthesis arcB SEQ ID NO: SEQ ID NO: nt. 49710-48706 10 Ornithine 625 626 of SEQ ID NO: carbamolytransferase; 681 (contig 14) arginine degradation uxuA SEQ ID NO: SEQ ID NO: nt. 840671-841855 3.1 Mannonate 627 628 of SEQ dehydratase; ID NO: 685 production of (contig 18) glyceraldehyde 3- phosphate dsbB SEQ ID NO: SEQ ID NO: nt. 388050-388583 2.6 Disulfide 629 630 of SEQ oxidoreductase; ID NO: 384 disulfide bond (contig 17) formation protein B ureH SEQ ID NO: SEQ ID NO: nt. 4452-5267 of 3.9 Urease accessory 631 632 SEQ ID NO: 680 protein (contig 13) licC SEQ ID NO: SEQ ID NO: nt. 355083-354382 2.3 Phosphocholine 633 634 of SEQ (ChoP) ID NO: 385 cytidylyltransferase (contig 18) HI1647 SEQ ID NO: SEQ ID NO: nt. 664017-664892 2.0 Putative pyridoxin 635 636 of SEQ biosynthesis protein; ID NO: 685 singlet oxygen (contig 18) resistance protein DNA ispZ SEQ ID NO: SEQ ID NO: nt. 4512-5069 of 2.5 Probable replication, 637 638 SEQ ID NO: 683 intracellular repair (contig 16) septation protein radC SEQ ID NO: SEQ ID NO: nt. 132695-132030 2.1 DNA repair protein 639 640 of SEQ ID NO: 683 (contig 16) mukF SEQ ID NO: SEQ ID NO: nt. 504549-503215 2.0 MukF protein 641 642 of SEQ homologue; ID NO: 685 remodeling of (contig 18) nucleiod structure Gene glpR SEQ ID NO: SEQ ID NO: nt. 72716-73483 2.8 Glycerol-3- regulation 643 644 of SEQ ID NO: phosphate regulon 682 (contig 15) repressor ihfB SEQ ID NO: SEQ ID NO: nt. 661004-660720 2.5 Integration host 645 646 of SEQ factor beta subunit ID NO: 685 (contig 18) argR SEQ ID NO: SEQ ID NO: nt. 178540-178085 2.7 Arginine repressor 647 648 of SEQ ID NO: 685 (contig 18) cspD SEQ ID NO: SEQ ID NO: nt. 435310-435528 2.1 Cold shock like 649 650 of SEQ protein; stress ID NO: 685 response protein (contig 18) Hypothetical HI1163 SEQ ID NO: SEQ ID NO: nt. 137202-134119 2.3 Conserved or 651 652 of SEQ hypothetical protein; unknown ID NO: 685 putative oxidase proteins (contig 18) HI1063 SEQ ID NO: SEQ ID NO: nt. 35158-34937 2.7 Hypothetical protein 653 654 of SEQ ID NO: 685 (contig 18) HI0665 SEQ ID NO: SEQ ID NO: nt. 17949-18980 2.8 Hypothetical protein 655 656 of SEQ ID NO: 679 (contig 12) HI1292 SEQ ID NO: SEQ ID NO: nt. 555002-555799 2.6 Hypothetical protein 657 658 of SEQ ID NO: 685 (contig 18)

Example 7 Identification of Virulence-Associated Genes

In many bacterial species, a subset of virulence-associated genes is regulated by errors in replication of short repeats. These repeats may be 5′ to a gene or in the coding sequence, and their presence is an indication of controlled expression of the gene, which indicates association with virulence. Addition or deletion of a repeat results in the expression or of lack of expression of the particular virulence determinant.

The NTHi H. influenzae strain 86-028NP contig set was queried for short oligonucleotide repeats. The region surrounding the repeats was analyzed to identify the gene(s) associated with the repeat. Table 5 lists the identified repeats and the ORF (identified by BLAST) associated with each repeat.

Further sequence analysis has identified the full length nucleotide sequence of the virulence-associated genes and the corresponding amino acid sequences encoded by the ORF. The derived amino acid sequences are highly homologous to the listed Genbank sequence.

TABLE 5 Location in Location in Full Length Amino 3-fold 8-fold Nucleotide Acid Genebank Repeat Contigs Contigs Sequence Sequence Accession No. SEQ ID 115 nt. 484533-483643 SEQ ID SEQ ID NP_439538.1 NO: 581 nt. 473-540 of NO: 659 NO: 660 of SEQ ID SEQ ID NO: 685 NO: 115 (contig 18) SEQ ID 377 nt. 416274-414910 SEQ ID SEQ ID P45217 NO: 582 nt. 546-597 of NO: 661 NO: 662 of SEQ ID NO: SEQ ID 685 (contig NO: 337 18) SEQ ID 505 nt. 414500-416614 SEQ ID SEQ ID AAK76425 NO: 583 nt. 310-393 of NO: 663 NO: 664 of SEQ ID NO: SEQ ID 684 (contig NO: 505 17) SEQ ID 508 nt. 506516-507913 SEQ ID SEQ ID NP_439520 NO: 584 nt. 2079-2120 of NO: 665 NO: 666 of SEQ ID NO: SEQ ID 685 (contig NO: 508 18) SEQ ID 518 nt. 354274-352406 SEQ ID SEQ ID NP_284893 NO: 585 nt. 758-789 of NO: 667 NO: 668 of SEQ ID NO: SEQ ID 684 (contig NO: 518 17) SEQ ID 543 nt. 347864-243236 SEQ ID SEQ ID AAA20524 NO: 586 nt. 1814-196 of NO: 669 NO: 670 of SEQ ID NO: SEQ ID 685 (contig NO: 543 18) SEQ ID 543 nt. 699709-704187 SEQ ID SEQ ID AAD56660 NO: 586 nt. 1814-196 of NO: 671 NO: 672 of SEQ ID NO: SEQ ID 685 (contig NO: 543 18) SEQ ID 567 nt. 85546-84689 SEQ ID SEQ ID ZP_00053190 NO: 587 nt. 13309-13320 of NO: 673 NO: 674 of SEQ ID NO: SEQ ID 681 (contig NO: 567 14)

Example 8 Identification of Unique NTHi Gene Sequences

Genes associated with NTHi virulence were also identified by comparing the level of expression of the gene when the NTHi bacterium was infecting a tissue verses the level of expression of the same gene when the NTHi was grown on artificial laboratory media. These novel genes were identified using the promoter trap techniques described above in Examples 4-6, and subsequently comparisons with the known Rd genome demonstrated these genes are unique to NTHi strain 86-028NP.

The DNA sequence identified using this screening procedure are set forth as SEQ ID NOS: 577-580. These sequences did not contain genes or gene fragments that have homologues in the H. influenzae Rd. genome sequence. Even though these are completely novel sequences, due to their expression level during NTHi infection in the chinchilla middle ear, it is likely that expression of these genes are involved in NTHi virulence.

Example 9 Complete Sequence Analysis of NTHi Strain 86-028NP

Library Construction:

Chromosomal DNA was prepared from strain 86-028NP using Puregene reagents (Gentra Systems, Minneapolis, Minn.). For the initial shotgun sequencing of the genome, 1 to 2 kb and 2 to 4 kb libraries of genomic DNA were constructed in pUC18 as previously described (Munson et al., Infect Immun 72:3002-10, 2004). For the scaffolding library, genomic DNA was manually sheared into a mean fragment size of 40 kb using a Hamilton syringe. After end repair, fragments were fractionated using a 0.7% low melting temperature agarose gel. Fragments larger than 30 kb were excised and an in-gel ligation to pEpiFOS-5 was performed. The ligation mixture recovered from the gel was packaged into Lambda phage, in vitro, and used to transfect EPI100 cells (Epicentre, Madison, Wis.).

Sequencing:

For the shotgun portion of the sequencing, cycle-sequencing reactions were run using PE Big-Dye™ terminators and universal primers (M13 forward and reverse) as previously described (Munson et al., Infect Immun 72:3002-10, 2004). To end-sequence the scaffolding library, plasmid was first purified using a R.E.A.L. Prep 96 Plasmid Kit (QIAGEN Inc., Valencia, Calif.), then amplified using a TempliPhi™ DNA Amplification Kit (Amersham Biosciences Corp., Piscataway, N.J.) before running reactions using PE Big-Dye™ terminators and pEpiFOS-5 forward and reverse sequencing primers (Epicentre, Madison, Wis.). The reactions for the clean-up portions of the project were run using PE Big-Dye™ terminators and custom primers (Integrated DNA Technologies, Coralville, Iowa). Excess dye terminators were removed with Sephadex G50 columns in 96-well format and sequence determined on either an ABI 3700 or an ABI 3100 capillary electrophoresis DNA sequencer (Applied Biosystems, Foster City, Calif.).

Genome Closure:

Paired end-sequences from the scaffolding library and PCR were used to order the contigs and to add sequence in areas of low sequence coverage. Paired custom primers (Integrated DNA Technologies, Coralville, Iowa) were designed to bind at the ends of each contig as well as regions flanking areas of low sequence coverage. The intervening regions were amplified with a standard PCR protocol as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (Third Edition). Cold Spring Harbor Laboratory Press., 2001 using Taq polymerase (Roche Diagnostics, Indianapolis, Ind.) and sequenced on both strands. Ribosomal RNA operons and the HMW gene clusters were completely sequenced using clones from the scaffolding library as templates.

Assembly:

Phred/Phrap was used for data assembly, employing the default assembly parameters (Ewing et al., Genome Res 8:186-94, 1998; Ewing et al., Genome Res 8:175-85, 1988; Gordon et al., Genome Res 8:195-202, 1998) as described in Munson et al., Infect Immun 72:3002-10, 2004. Assemblies were checked using the paired-end sequence data from 507 clones using the Seqman II program from the DNASTAR suite.

Data Analyses:

Coding regions were identified using Glimmer2 (v2.13) trained on the set of 1178 longest ORFs identified by the Glimmer2 long-orfs program (Delcher et al., Nucleic Acids Res 27:4636-41, 1999). Automated annotation by similarity was done by searching the Glimmer ORF set against the strain Rd proteome, the SwissProt database, the NCBI COGs database, and the KEGG database. The strain Rd database was compared bi-directionally with the strain 86-028NP ORF set using tricross to determine high-confidence regions of similarity and to produce the dotplot comparison of genome organization Ray et al., Bioinformatics 17:1105-12, 2001).

The automatically predicted annotation information was further manually curated using Artemis (Rodriguez et al., Infect Immun 71:1635-42, 2003) for visualization and demarcation of genomic regions of interest, and a custom FileMaker Pro database was generated which was then used to apply manual revisions and archive data related to the functional assignment. FASTA analyses were used for the primary automated comparisons. The strong synteny between the strain 80-028NP and strain Rd genomes allowed assignment of a function to the majority of the genes automatically, with similarity held to 90% or better at the amino acid level for matching. The near one-to-one mapping from the strain 86-028NP genome to the strain Rd genome was confirmed by assembly of the strain Rd ORFs onto the strain 86-028NP genome sequence, and the reverse assembly of the strain 86-028NP ORFs onto the Rd genome, using the SeqMan program with the assembly criteria of 80% identity at the nucleotide level.

Manual BLAST analyses were used to explore the potential function of ORFs that did not show strong similarity to known genes. Manual curation of the automatic assignments was carried out to conform annotations to the current literature and repair the few places where the automated algorithm was easily led astray (notably the HMW gene clusters, the hemoglobin-binding proteins and the hsd gene clusters, whose high family similarity confounds automated assignment).

The tRNA genes were identified by tRNAscan-SE v1.11 (Lowe et al., Nucleic Acids Res 25:955-64, 1997). The rRNA operons were identified based on 16, 23 and 5S rRNA similarity with strain Rd, and the CLUSTALW alignment of the neighborhoods containing these genes to determine the boundaries of the semi-conserved regions.

The complete genomic DNA sequence of the H. influennzae, strain 86-028NP, is set out as SEQ ID NO: 772. The open reading frames within the genomic DNA are set out as SEQ ID NOS: 773-2593 and are described in Table 6. The resulting gene products are set out as SEQ ID NOS: 2581-4414 and are described in Table 7. The “c” preceding the nucleotides of the open reading frames indicates the open reading frame is complementarty to the 5′ to 3′ sequence set out as SEQ ID NO: 722. The unique NTHi genes are marked in Table 6. The genome sequence will be available from GenBank as Accession number CP000057, which is incorporated by reference herein in its entirety.

The following open reading frames are defined as pseudogenes in Table 6 because these nucleotide sequences contain a frameshift or a stop codon within the sequence: SEQ ID NO: 822 (kdgK), SEQ ID NO: 928, SEQ ID NO: 994 (bolA), SEQ ID NO: 276 (adhC) SEQ ID NO: 1102 (dusC), SEQ ID NO: 1121 (merP), SEQ ID NO: 1135, SEQ ID NO:1236, SEQ ID NO: 1254, SEQ ID NO: 1376 (lic2C), SEQ ID NO: 1431 (hgpD), SEQ ID NO: 1502, SEQ ID NO:1505 (ppx), SEQ ID NO: 1523 (hgpC), SEQ ID NO: 1585 (lex2A), SEQ ID NO: 1637 SEQ ID NO: 1713, SEQ ID NO: 1856 (mod), SEQ ID NO: 1899, SEQ ID NO: 2006, SEQ ID NO: 2080, SEQ ID NO: 2155, SEQ ID NO: 2202, SEQ ID NO:2257, SEQ ID NO: 2331, SEQ ID NO:2345, SEQ ID NO: 2365, SEQ ID NO: 2555 (metE) and SEQ ID NO:2563 (pmi). The hgpD, hgpC, lex2A and NTHI1769 genes contain contingency repeats that cause the sequence to shift in and out of the reading frame and the resulting amino acid sequences (SEQ ID NOS: 3242, 3332 and 4142) are partial translation. These contingency repeats regulate gene expression in the H. influenzae and therefore the genes encoding these proteins will be correctly translated after insertion or deletion of 1 or more repeats

TABLE 6 NTHi Open Reading Frames and Gene Poducts Unique Gene to NTHi SEQ CDS Name 86-0268 Product Name ID NO:   2-1021 gapA glyceraldehyde 3-phosphate dehydrogenase 773 1191-3014 — putative long-chain-fatty-acid--CoA ligase 774 C 3051-3839 — conserved hypothetical protein 775 C 3855-4319 — conserved hypothetical protein 776 C 4581-5393 fdhD FdhD protein homolog 777 5664-8750 fdxG formate dehydrogenase major subunit 778 8752-9690 fdxH formate dehydrogenase, iron-sulfur subunit 779  9683-10399 fdxI formate dehydrogenase, cytochrome B556 780 subunit 10469 . . . 11377 fdhE FdhE homolog 781 C 11421 . . . 11861 rimI ribosomal-protein-alanine acetyltransferase 782 C 11864 . . . 12268 holD DNA polymerase III, psi subunit 783 12374 . . . 13366 rsmC ribosomal RNA small subunit 784 methyltransferase C C 13434 . . . 14342 era GTP-binding protein era homolog 785 C 14339 . . . 15022 rnc ribonuclease III 786 C 15024 . . . 16073 lepB Signal peptidase I 787 C 16082 . . . 17878 lepA GTP-binding protein LepA 788 C 18047 . . . 18430 — conserved hypothetical acid-induced glycyl 789 radical enzyme 18688 . . . 19347 ung uracil-DNA glycosylase 790 C 19417 . . . 20841 — conserved hypothetical protein 791 C 21224 . . . 22663 — conserved hypothetical protein 792 C 22665 . . . 24062 citG CitXG 793 C 24243 . . . 25745 citF citrate lyase alpha chain 794 C 25760 . . . 26635 citE citrate lyase beta chain 795 C 26632 . . . 26919 citD citrate lyase acyl carrier protein 796 C 26957 . . . 27964 citC [citrate [pro-3S]-lyase] ligase 797 C 28215 . . . 29177 lipA Lipoic acid synthetase 798 C 29231 . . . 29869 lipB lipoate-protein ligase B 799 C 29871 . . . 30149 — conserved hypothetical protein 800 C 30205 . . . 31386 dacA penicillin-binding protein 5 precursor 801 C 31402 . . . 32265 rlpA RlpA-like protein 802 C 32318 . . . 33433 mrdB Rod shape-determining protein RodA 803 C 33423 . . . 35378 mrdA penicillin-binding protein 2 804 C 35400 . . . 35867 — conserved hypothetical protein 805 C 35921 . . . 36229 — conserved hypothetical protein 806 C 36354 . . . 38009 — conserved hypothetical membrane protein 807 38215 . . . 39993 — conserved hypothetical ABC transporter ATP- 808 binding protein 40042 . . . 41178 mreB rod shape-determining protein MreB 809 41258 . . . 42313 mreC rod shape-determining protein MreC 810 42313 . . . 42801 mreD rod shape-determining protein MreD 811 C 42852 . . . 43628 — conserved hypothetical protein 812 C 43638 . . . 44441 xthA exodeoxyribonuclease III 813 C 44489 . . . 45163 rluA2 conserved hypothetical pseudouridine synthase 814 C 45163 . . . 46227 — conserved hypothetical membrane protein 815 C 46287 . . . 46949 — conserved hypothetical FtsH-interacting 816 integral membrane protein C 47276 . . . 47830 — conserved hypothetical protein 817 47897 . . . 48259 phnA PhnA homolog 818 C 48312 . . . 48950 eda KHG/KDPG aldolase 819 C 48959 . . . 50362 uxuC ✓ uronate isomerase 820 C 50372 . . . 51229 — putative oxidoreductase 821 C 51229 . . . 52172 kdgK pseudogene for 2-dehydro-3- 822 deoxygluconokinase C 52192 . . . 53463 — putative TRAP-type C4-dicarboxylate transport 823 system, large permease component C 53473 . . . 53970 — putative TRAP-type C4-dicarboxylate transport 824 system, small permease component C 54009 . . . 54995 — putative TRAP-type C4-dicarboxylate transport 825 system, periplasmic component C 55022 . . . 56050 — conserved hypothetical zinc-type alcohol 826 dehydrogenase-like protein 56160 . . . 56960 uxuR Uxu operon regulator 827 56988 . . . 58172 uxuA mannonate dehydratase 828 C 58225 . . . 58938 — putative membrane protein TerC 829 C 59036 . . . 60865 uvrC UvrABC system protein C 830 C 60867 . . . 61631 kdsB 3-deoxy-D-manno-octulosonic acid 831 cytidylyltransferase C 61702 . . . 62700 lpxK tetraacyldisaccharide 4′-kinase 832 C 62773 . . . 64536 msbA lipid A export ATP-binding protein MsbA 833 C 64577 . . . 66943 rec2 recombination protein 2 834 67202 . . . 67639 dksA DnaK suppressor protein 835 67887 . . . 69245 pcnB probable poly polymerase 836 69254 . . . 69736 folK 2-amino-4-hydroxy-6-hydroxymethyl- 837 dihydropteridine pyrophosphokinase 69812 . . . 70288 — conserved hypothetical protein 838 70296 . . . 71594 amiB probable N-acetylmuramoyl-L-alanine amidase 839 AmiB precursor 71595 . . . 73484 mutL DNA mismatch repair protein MutL 840 73492 . . . 74427 miaA tRNA delta-isopentenylpyrophosphate 841 transferase 74433 . . . 77378 glnE glutamate-ammonia-ligase adenylyltransferase 842 C 77463 . . . 79139 recN DNA repair protein RecN 843 C 79251 . . . 80069 ppnK probable inorganic polyphosphate/ATP-NAD 844 kinase 80133 . . . 80837 grpE grpE 845 C 80850 . . . 81194 — conserved hypothetical protein 846 C 81187 . . . 81627 — conserved hypothetical protein 847 81908 . . . 84031 nrdD anaerobic ribonucleoside-triphosphate 848 reductase 84150 . . . 85010 tesB Acyl-CoA thioesterase II 849 C 85171 . . . 86550 cysS cysteinyl-tRNA synthetase 850 86653 . . . 87162 ppiB peptidyl-prolyl cis-trans isomerase B 851 87166 . . . 87597 — conserved hypothetical protein 852 87739 . . . 88527 — putative deoxyribonuclease 853 88589 . . . 88846 — ✓ hypothetical protein 854 88857 . . . 89132 — ✓ hypothetical protein 855 C 89182 . . . 89505 trxA thioredoxin 856 C 89625 . . . 90620 ddh 2-hydroxyacid dehydrogenase homolog 857 C 90633 . . . 91778 metB cystathionine gamma-synthase 858 92334 . . . 93170 — ✓ predicted ATPase involved in chromosome 859 partitioning 93172 . . . 94527 dnaB2 ✓ replicative DNA helicase 860 94520 . . . 96220 — ✓ conserved hypothetical protein 861 96220 . . . 96771 — ✓ conserved hypothetical protein 862 96921 . . . 98144 — ✓ conserved hypothetical protein 863 98441 . . . 99196 — ✓ conserved hypothetical protein 864 99196 . . . 99684 — ✓ conserved hypothetical protein 865 99930 . . . 100352 ssb2 ✓ Single-strand binding protein 866 100379 . . . 100897 — ✓ conserved putative lipoprotein 867 C 101113 . . . 101670 — ✓ 1conserved hypothetical protein 868 101746 . . . 103806 topB2 ✓ DNA topoisomerase III 869 104691 . . . 105119 — ✓ conserved hypothetical protein 870 105131 . . . 105223 — hypothetical protein 871 105234 . . . 105875 — ✓ conserved hypothetical protein 872 105910 . . . 106317 — ✓ hypothetical protein 873 106418 . . . 106888 radC2 ✓ putative DNA repair radC-like protein 874 106981 . . . 107577 — ✓ conserved hypothetical protein 875 107635 . . . 108399 — ✓ conserved hypothetical protein 876 108498 . . . 109127 pilL ✓ conserved putative lipoprotein 877 109131 . . . 109871 — ✓ conserved putative exported protein 878 109850 . . . 110608 — ✓ conserved putative exported protein 879 110624 . . . 111130 — ✓ conserved putative exported protein 880 111127 . . . 113376 — ✓ conserved hypothetical protein 881 113695 . . . 114381 — ✓ conserved putative membrane protein 882 114512 . . . 114862 — ✓ conserved putative membrane protein 883 115131 . . . 115532 — ✓ conserved putative membrane protein 884 115552 . . . 115923 — ✓ conserved putative membrane protein 885 115935 . . . 116579 — ✓ conserved putative exported protein 886 116579 . . . 117448 — ✓ conserved hypothetical protein 887 117459 . . . 118859 — ✓ conserved putative exported protein 888 118869 . . . 119276 — ✓ conserved putative lipoprotein 889 119292 . . . 122162 — ✓ conserved hypothetical protein 890 122170 . . . 122577 — ✓ conserved hypothetical protein 891 122587 . . . 122910 — ✓ conserved hypothetical membrane protein 892 122919 . . . 124412 — ✓ conserved putative membrane protein 893 C 124468 . . . 124797 — ✓ conserved hypothetical membrane protein 894 C 124955 . . . 127960 tnpA ✓ transposon Tn3 transposase 895 126925 . . . 127041 — ✓ hypothetical protein 896 128088 . . . 128726 tnpR ✓ transposon Tn3 resolvase 897 128751 . . . 128930 — ✓ hypothetical protein 898 C 128975 . . . 129382 — ✓ hypothetical exported protein 899 C 129398 . . . 131398 — ✓ conserved putative exported protein 900 C 131413 . . . 132354 — ✓ conserved putative exported protein 901 C 132351 . . . 132794 — ✓ conserved hypothetical protein 902 133106 . . . 133525 — ✓ conserved hypothetical membrane protein 903 133610 . . . 133843 — ✓ conserved hypothetical protein 904 133846 . . . 134094 — ✓ hypothetical protein 905 134098 . . . 134256 — ✓ hypothetical protein 906 134387 . . . 135340 traC ✓ conserved putative antirestriction protein 907 135444 . . . 136205 — ✓ possible type I restriction enzyme M subunit 908 136431 . . . 136997 — ✓ hypothetical protein 909 137037 . . . 137411 — ✓ conserved hypothetical protein 910 137401 . . . 138078 — ✓ conserved hypothetical protein 911 138056 . . . 138673 — ✓ conserved hypothetical protein 912 C 138937 . . . 139536 — ✓ resolvase/integrase-like protein 913 C 139551 . . . 139742 — ✓ hypothetical protein 914 C 140211 . . . 140465 — ✓ hypothetical protein 915 141656 . . . 142369 — ✓ conserved hypothetical protein 916 143089 . . . 144342 — ✓ hypothetical protein 917 144485 . . . 146401 — ✓ conserved hypothetical protein 918 146462 . . . 147304 — ✓ putative site-specific recombinase 919 C 147954 . . . 149231 thrC threonine synthase 920 C 149274 . . . 150218 thrB homoserine kinase 921 C 150231 . . . 152678 thrA aspartokinase/homoserine dehydrogenase 922 153002 . . . 153715 — conserved hypothetical protein 923 153755 . . . 154891 grk glycerate kinase 924 C 154900 . . . 156159 — conserved hypothetical protein 925 C 156281 . . . 157387 — conserved hypothetical protein 926 C 157589 . . . 157909 — conserved hypothetical protein 927 157962 . . . 158171 — pseudogene for glycerol-3-phosphate regulon 928 repressor C 158260 . . . 159015 — conserved hypothetical protein 929 159216 . . . 159497 — conserved hypothetical protein 930 159649 . . . 160647 hitA iron-utilization periplasmic protein hFbpA 931 160765 . . . 162285 hitB iron(III)-transport system permease protein 932 hFbpB 162287 . . . 163342 hitC iron-utilization ATP-binding protein hFbpC 933 C 163377 . . . 164057 — putative D-alanyl-D-alanine carboxypeptidase 934 C 164059 . . . 165192 dapE succinyl-diaminopimelate desuccinylase 935 C 165220 . . . 165564 — conserved hypothetical protein 936 C 165643 . . . 167538 — chaperone protein HtpG 937 167750 . . . 168589 — conserved hypothetical NIF3-like protein 938 168763 . . . 171135 hsdM1 ✓ putative type I restriction-modification system, 939 methyltransferase subunit 171318 . . . 172727 — ✓ predicted transcriptional regulator containing 940 an HTH domain 172899 . . . 174149 hsdS1 ✓ putative type I site-specific restriction- 941 modification system, S subunit 174249 . . . 177275 hsdR1 ✓ putative type I site-specific restriction- 942 modification system, R subunit C 177500 . . . 178879 ffh Signal recognition particle protein 943 179130 . . . 180422 corB putative Mg2+ and Co2+ transporter 944 180443 . . . 181321 — conserved hypothetical protein 945 181323 . . . 181802 — ✓ conserved hypothetical protein 946 181892 . . . 182233 — conserved hypothetical protein 947 182400 . . . 183689 serS Seryl-tRNA synthetase 948 184024 . . . 184653 gst glutathione S-transferase 949 C 184665 . . . 184961 — conserved hypothetical protein 950 185014 . . . 187251 hemR hemin receptor 951 C 194299 . . . 196434 — possible SAM-dependent methyltransferase 952 196827 . . . 197936 mltA membrane-bound lytic murein transglycosylase A 953 precursor 197936 . . . 198706 — conserved hypothetical protein 954 198884 . . . 199885 znuA high-affinity zinc uptake system protein ZnuA 955 199935 . . . 200441 — conserved hypothetical protein 956 C 200554 . . . 201912 mpl UDP-N-acetylmuramate:L-alanyl-gamma-D- 957 glutamyl-meso-diaminopimelate ligase C 202297 . . . 203484 metC cystathionine beta-lyase 958 203615 . . . 204223 tsaA ✓ probable peroxiredoxin 959 204324 . . . 204881 pgsA CDP-diacylglycerol--glycerol-3-phosphate 3- 960 phosphatidyltransferase C 205445 . . . 205975 ppa inorganic pyrophosphatase 961 206209 . . . 207525 — conserved hypothetical protein 962 207748 . . . 208389 udk uridine kinase 963 208398 . . . 208985 dcd deoxycytidine triphosphate deaminase 964 208989 . . . 210164 — conserved hypothetical protein 965 210164 . . . 211372 — probable sugar efflux transporter 966 C 211433 . . . 212947 engA GTP-binding protein EngA 967 C 213629 . . . 214396 dnaQ DNA polymerase III, epsilon chain 968 214457 . . . 214921 rnhA ribonuclease HI 969 215187 . . . 216284 omP2 Outer membrane protein P2 precursor 970 C 216481 . . . 217626 nagA N-acetylglucosamine-6-phosphate deacetylase 971 C 217763 . . . 218575 nagB glucosamine-6-phosphate deaminase 972 C 218911 . . . 219792 nanA N-acetylneuraminate lyase 973 C 219803 . . . 220669 — putative HTH-type transcriptional regulator 974 C 220662 . . . 221564 nanK putative N-acetylmannosamine kinase 975 C 221609 . . . 222295 nanE putative N-acetylmannosamine-6-phosphate 976 2-epimerase 222645 . . . 223634 — putative sialic acid transporter, TRAP-type 977 C4-dicarboxylate transport system, periplasmic component 223699 . . . 225549 siaT putative sialic acid transporter, TRAP-type 978 C4-dicarboxylate transport system, large permease component 225713 . . . 226852 — conserved hypothetical protein 979 227014 . . . 227448 — putative protein-S-isoprenylcysteine 980 methyltransferase C 227490 . . . 228377 hflC HflC 981 C 228377 . . . 229609 hflK HhflK 982 C 229719 . . . 230426 — putative 4′-phosphopantetheinyl transferase 983 C 230486 . . . 231817 dcuB2 anaerobic C4-dicarboxylate transporter DcuB 984 C 232021 . . . 232251 acpP acyl carrier protein 985 C 232511 . . . 233239 fabG 3-oxoacyl-[acyl-carrier protein] reductase 986 C 233256 . . . 234194 fabD malonyl CoA-acyl carrier protein transacylase 987 C 234694 . . . 235644 fabH 3-oxoacyl-[acyl-carrier-protein] synthase III 988 C 235830 . . . 236000 rpmF 50S ribosomal protein L32 989 C 236023 . . . 236547 — conserved hypothetical protein 990 236704 . . . 237576 psd phosphatidylserine decarboxylase proenzyme 991 C 237685 . . . 239055 gor glutathione reductase 992 C 239271 . . . 239870 — conserved hypothetical lipoprotein 993 239964 . . . 240275 bolA Pseudogene for BolA homolog 994 240566 . . . 241909 nqrA Na(+)-translocating NADH-quinone reductase 995 subunit A 241912 . . . 243147 nqrB Na(+)-translocating NADH-quinone reductase 996 subunit B 243140 . . . 243874 nqrC Na(+)-translocating NADH-quinone reductase 997 subunit C 243874 . . . 244500 nqrD Na(+)-translocating NADH-quinone reductase 998 subunit D 244504 . . . 245100 nqrE Na(+)-translocating NADH-quinone reductase 999 subunit E 245113 . . . 246348 nqrF Na(+)-translocating NADH-quinone reductase 1000 subunit F 246492 . . . 247532 apbE thiamine biosynthesis lipoprotein ApbE 1001 247535 . . . 247795 — conserved hypothetical protein 1002 247870 . . . 249063 trmU probable tRNA (5-methylaminomethyl-2- 1003 thiouridylate)-methyltransferase C 249107 . . . 249841 — conserved hypothetical protein 1004 C 249843 . . . 250817 rluD ribosomal large subunit pseudouridine synthase D 1005 250925 . . . 251713 — conserved hypothetical lipoprotein 1006 C 251792 . . . 252274 — conserved hypothetical protein 1007 C 252306 . . . 253046 pflA pyruvate formate-lyase 1 activating enzyme 1008 253171 . . . 255489 pflB formate acetyltransferase 1009 C 255521 . . . 256375 focA probable formate transporter 1010 256880 . . . 257794 — conserved hypothetical protein 1011 257973 . . . 259418 — putative Na+/alanine symporter 1012 C 259603 . . . 260430 — conserved hypothetical protein 1013 C 260439 . . . 261575 adhC pseudogene for putative alcohol dehydrogenase 1014 class III 261698 . . . 262105 — putative HTH-type transcriptional regulator 1015 262263 . . . 262550 tatA Sec-independent protein translocase protein 1016 TatA/E 262554 . . . 263114 tatB Sec-independent protein translocase protein 1017 TatB 263124 . . . 263894 tatC Sec-independent protein translocase protein 1018 TatC 264255 . . . 265604 gdhA NADP-specific glutamate dehydrogenase 1019 C 265712 . . . 266152 fur Ferric uptake regulation protein 1020 C 266181 . . . 266705 fldA flavodoxin 1021 C 267655 . . . 268260 seqA seqA 1022 266802 . . . 267665 — putative esterase/lipase 1023 268263 . . . 269621 menE O-succinylbenzoate--CoA ligase 1024 269649 . . . 272987 — putative small-conductance mechanosensitive 1025 channel 272997 . . . 274070 aroC chorismate synthase 1026 274243 . . . 275103 mepA penicillin-insensitive murein endopeptidase 1027 275121 . . . 275888 — conserved hypothetical protein 1028 275935 . . . 276891 msbB lipid A biosynthesis (KDO)2-(lauroyl)-lipid 1029 IVA acyltransferase 277018 . . . 277989 selD selenide, water dikinase 1030 C 278098 . . . 278448 rplsS 50S ribosomal protein L19 1031 C 278485 . . . 279225 trmD tRNA (guanine-N(1)-)-methyltransferase 1032 C 279263 . . . 279790 rimM 16S rRNA processing protein RimM 1033 C 279835 . . . 280083 rspP 30S ribosomal protein S16 1034 280363 . . . 281148 — conserved hypothetical protein 1035 281171 . . . 282982 nadN NAD nucleotidase 1036 283267 . . . 283809 aroK shikimate kinase 1037 283829 . . . 284917 aroB 3-dehydroquinate synthase 1038 284919 . . . 285779 dam DNA adenine methylase 1039 286744 . . . 286851 — conserved hypothetical protein 1040 C 286870 . . . 287595 pgpB phosphatidylglycerophosphatase B 1041 287641 . . . 288315 ribA GTP cyclohydrolase II 1042 C 288631 . . . 290175 — putative ABC-type oligopeptide transport 1043 system, periplasmic component C 290168 . . . 290530 — conserved hypothetical protein 1044 290649 . . . 292694 prlc oligopeptidase A 1045 292926 . . . 294470 Hsdm2 putative type I restriction-modification system 1046 methyltransferase subunit 294555 . . . 295727 Hsds2 putative type I restriction-modification system 1047 specificity protein 295729 . . . 296883 prrC ✓ putative anticodon nuclease 1048 296886 . . . 297884 — ✓ conserved hypothetical DNA binding protein 1049 297895 . . . 300978 Hsdr2 putative type I restriction-modification system 1050 C 301125 . . . 301754 — conserved hypothetical protein 1051 C 301968 . . . 303809 arcb aerobic respiration control sensor protein ArcB 1052 C 303878 . . . 304522 — predicted uracil-DNA glycosylase 1053 C 310574 . . . 311482 bira BirA bifunctional protein 1054 311609 . . . 313075 guab inosine-5′-monophosphate dehydrogenase 1055 313185 . . . 314756 guaa glutamine-hydrolyzing GMP synthase 1056 C 314818 . . . 315720 — conserved hypothetical protein 1057 315783 . . . 316289 — putative transcriptional regulator 1058 316328 . . . 317530 nhaa Na(+)/H(+) antiporter 1 1059 317718 . . . 319028 brnq branched-chain amino acid transport system 1060 carrier protein C 319136 . . . 319603 — conserved hypothetical protein 1061 C 319630 . . . 320076 — conserved hypothetical protein 1062 320349 . . . 322478 pnp polyribonucleotide nucleotidyltransferase 1063 322560 . . . 323504 nlpL lipoprotein NlpI 1064 323624 . . . 325465 deaD Cold-shock DEAD-box protein A homolog 1065 325643 . . . 327031 — predicted soluble lytic transglycosylase fused 1066 to an ABC-type amino acid-binding protein 327036 . . . 327188 — conserved hypothetical protein 1067 C 327322 . . . 327618 — conserved hypothetical protein 1068 C 327675 . . . 327884 — conserved hypothetical protein 1069 C 327952 . . . 328302 arsC putative arsenate reductase 1070 328381 . . . 329430 perM putative permease PerM homolog 1071 C 329544 . . . 330521 secF protein-export membrane protein SecF 1072 C 330529 . . . 332379 secD protein-export membrane protein SecD 1073 C 332449 . . . 332742 — conserved hypothetical preprotein translocase 1074 subunit YajC C 332850 . . . 333071 — predicted redox protein, regulator of disulfide 1075 bond formation C 333068 . . . 333586 — conserved hypothetical protein 1076 C 333651 . . . 334799 tgt queuine tRNA-ribosyltransferase 1077 C 335144 . . . 336235 quea S-adenosylmethionine:tRNA ribosyltransferase- 1078 isomerase C 336359 . . . 336805 — conserved hypothetical protein 1079 C 336948 . . . 341126 hap ✓ adhesion and penetration protein Hap 1080 C 341444 . . . 344275 uvra UvrABC system protein A 1081 344428 . . . 344934 ssb Single-strand binding protein 1082 C 345112 . . . 345906 tonb TonB 1083 345916 . . . 346359 exbD transport protein ExbD 1084 346363 . . . 346815 exbB transport protein ExbB 1085 346984 . . . 347451 bcp bacterioferritin comigratory protein 1086 347552 . . . 348448 dapA dihydrodipicolinate synthase 1087 348559 . . . 349188 — conserved hypothetical lipoprotein 1088 349378 . . . 349701 — conserved hypothetical protein 1089 C 350415 . . . 351353 lgtC UDP-galactose--lipooligosaccharide 1090 galactosyltransferase C 351421 . . . 352008 orfM predicted xanthosine triphosphate 1091 pyrophosphatase C 352020 . . . 352745 kdkA 3-deoxy-D-manno-octulosonic acid kinase 1092 352822 . . . 353865 opsX ADP-heptose--lipooligosaccharide 1093 heptosyltransferase I 354187 . . . 356316 hxuC heme/hemopexin-binding protein C 1094 356392 . . . 358089 hxuB heme/hemopexin-binding protein B 1095 358101 . . . 360866 hxuA heme/hemopexin-binding protein A 1096 C 360962 . . . 361318 folB dihydroneopterin aldolase 1097 361401 . . . 362000 — conserved hypothetical protein 1098 362042 . . . 363745 narQ sensor protein NarQ 1099 363755 . . . 364780 murB UDP-N-acetylenolpyruvoylglucosamine 1100 reductase 364906 . . . 365751 rpoH RNA polymerase sigma-32 factor 1101 C 365791 . . . 366218 dusC ✓ pseudogene for tRNA-dihydrouridine synthase 1102 C- C 366218 . . . 367084 djlA DnaJ-like protein DjlA 1103 C 367163 . . . 367804 pyrE orotate phosphoribosyltransferase 1104 C 367828 . . . 368544 rph ribonuclease PH 1105 C 368845 . . . 370287 gltX glutamyl-tRNA synthetase 1106 371037 . . . 372692 Lpt6 PE-tn-6--lipooligosaccharide 1107 phosphorylethanolamine transferase C 372710 . . . 373519 rbn tRNA processing ribonuclease BN 1108 C 373516 . . . 374001 — conserved hypothetical protein 1109 C 374004 . . . 374666 — conserved hypothetical protein 1110 374881 . . . 375660 udp uridine phosphorylase 1111 C 375886 . . . 377202 — conserved hypothetical metabolite transport 1112 protein C 377258 . . . 378001 — conserved hypothetical protein 1113 C 378062 . . . 379768 mend menaquinone biosynthesis protein MenD 1114 C 379784 . . . 381055 menf menaquinone-specific isochorismate synthase 1115 381212 . . . 382426 Aspc3 probable aspartate aminotransferase 1116 382543 . . . 383799 mtr tryptophan-specific transport protein 1117 383908 . . . 385275 sdaa L-serine dehydratase 1118 C 385310 . . . 386548 sdac Serine transporter 1119 C 386771 . . . 388939 — probable cation-transporting ATPase 1120 C 388914 . . . 389702 merP pseudogene for probable inorganic ion transport 1121 protein 389779 . . . 390165 — ✓ probable heavy metal dependent transcriptional 1122 regulator C 390179 . . . 390496 metJ Met repressor 1123 390740 . . . 392002 rho transcription termination factor rho 1124 C 392056 . . . 392748 pilD putative type 4 prepilin-like protein specific 1125 leader peptidase C 392745 . . . 393965 pilC putative type IV pilin secretion protein 1126 C 393962 . . . 395356 pilB putative type IV pilin secretion protein 1127 C 395353 . . . 395802 pilA Type IV pilin subunit protein 1128 395917 . . . 396477 ampD ampD 1129 397099 . . . 397998 corC magnesium and cobalt efflux protein CorC 1130 397982 . . . 399550 cutE apolipoprotein N-acyltransferase 1131 399600 . . . 400337 — conserved hypothetical RNA methyltransferase 1132 400334 . . . 400894 — conserved hypothetical protein 1133 400894 . . . 401313 ruvX putative holliday junction resolvase 1134 C 401363 . . . 402288 — pseudogene for recombination associated 1135 protein RdgC, 402483 . . . 403694 — ✓ prophage CP4-57-like integrase 1136 403731 . . . 404165 — ✓ hypothetical protein 1137 404278 . . . 404700 — ✓ hypothetical protein 1138 404709 . . . 405482 — ✓ hypothetical protein 1139 406017 . . . 406208 — ✓ hypothetical protein 1140 406257 . . . 406409 — ✓ hypothetical protein 1141 C 406576 . . . 407466 — ✓ hypothetical protein 1142 408127 . . . 408942 proC pyrroline-5-carboxylate reductase 1143 408942 . . . 410108 hcaT probable 3-phenylpropionic acid transporter 1144 410139 . . . 411032 xerD Site-specific recombinase XerD 1145 411205 . . . 411492 — conserved hypothetical protein 1146 C 411544 . . . 412551 ruvB holliday junction DNA helicase RuvB 1147 C 412559-413173 ruvA holliday junction DNA helicase RuvA 1148 C 413236-413808 ruvC holliday junction DNA helicase RuvC 1149 C 413885-414595 — conserved hypothetical protein 1150 C 414607-415080 ntpA dATP pyrophosphohydrolase 1151 C 415102-416868 aspS Aspartyl-tRNA synthetase 1152 417087-417605 — conserved hypothetical protein 1153 417658-418383 — conserved hypothetical protein 1154 418476-418712 — conserved hypothetical protein 1155 418709-419113 — Predicted nucleic acid-binding domain, 1156 containsPIN domain 419180-419587 gloA Lactoylglutathione lyase 1157 419661-420350 Rnt Ribonuclease T 1158 420664-422016 — conserved hypothetical protein 1159 422049-422636 — Predicted primosomal replication protein N 1160 C 422987-423553 Efp Elongation factor P 1161 423591-424607 — Predicted lysine 2,3-aminomutase 1162 424706-426001 oapA Opacity associated protein OapA 1163 426060-426464 oapB Opacity associated protein OapB 1164 426467-427177 recO DNA repair protein RecO 1165 427177-428493 rumA 23S rRNA (uracil-5--)methyltransferase RumA 1166 428571-430802 relA GTP pyrophosphokinase 1167 430817-431173 dgkA Diacylglycerol kinase 1168 431256-431849 Mog Molybdopterin biosynthesis mog protein 1169 431851-432189 glnB Nitrogen regulatory protein P-II 1170 432189-433235 — conserved hypothetical protein 1171 C 433277-435469 priA Prismosomal protein N 1172 435554-436294 trmB tRNA (guanine-N(7)-)-methyltransferase 1173 436379-436723 — conserved hypothetical protein 1174 436929-437459 napF Ferredoxin-type protein NapF 1175 437452-437733 napD NapD 1176 437755-440253 napA Periplasmic nitrate reductase 1177 440307-441146 napG Ferredoxin-type protein NapG 1178 441146-442009 napH Ferredoxin-type protein NapH 1179 442006-442458 napB Diheme cytochrome C NapB 1180 442473-443075 napC Cytochrome C-type protein NapC 1181 C 443235-443879 Adk Adenylate kinase 1182 C 443964-445241 Lic3c Putative integral membrane signal transducer 1183 protein C 445383-446399 galE UDP-glucose 4-epimerase 1184 C 446572-447534 Lic3A CMP-Neu5Ac--lipooligosaccharide alpha 2-3 1185 sialytransferase 447933-448655 — Putative ABC-type 1186 nitrate/sulfonate/bicarbonate transport system, ATPase component 448652-449389 — Putative ABC-type 1187 nitrate/sulfonate/bicarbonate transport system, permease component 449411-450355 — Putative ABC-type 1188 nitrate/sulfonate/bicarbonate transport system, periplasmic component 450365-451012 — Putative transcription activator 1189 C 451107-451922 hfeD Putative ABC-type chelated iron transport 1190 system, permease component C 451915-452763 hfeC Putative ABC-type chelated iron transport 1191 system, permease component C 452767-453687 hfeB Putative ABC-type chelated iron transport 1192 system, ATPase component C 453687-454568 hfeA Putative periplasmic chelated iron binding 1193 protein 454874-455338 — ✓ Hypothetical protein 1194 C 455430-456308 pbpG Penicillin-binding protein 7 homolog precursos 1195 456533-457705 — Predicted Fe—S-cluster redox enzyme 1196 457805-458344 — Conserved hypothetical protein 1197 458416-459327 — Conserved hypothetical transcriptional 1198 regulator with an N-terminal xre-type HTH domain 459336-460442 gcpE 4-hydroxy-2-methyl-2-(E)-butenyl 4- 1199 diphosphate synthase 460452-461723 hisS Histidyl-tRNA synthetase 1200 461741-462355 — Conserved hypothetical protein 1201 C 462406 . . . 462600 — conserved hypothetical protein 1202 C 462600-462941 Fdx Feerdoxin, 2Fe—S 1203 C 462981-464840 hscA Chaperone protein HscA 1204 C 464859-465545 — Conserved hypothetical protein 1205 C 465596-466120 hscB Co-chaperone protein HscB 1206 C 466133-466456 — Conserved hypothetical protein 1207 C 466514-466894 nifU NifU-like protein 1208 C 466954-468174 nifS2 Cysteine sedulferase 1209 C 468235-468687 — Predicted transcriptional regulator 1210 C 468740-469465 — Hypothetical tRNA/rRNA methyltransferase 1211 C 469905-470366 Pal Outer membrane protein P6 precursor 1212 C 470390-471673 tolB TolB 1213 C 471715-472941 tolA TolA 1214 C 472957-473376 tolR TolR 1215 C 473447-474133 tolQ TolQ 1216 C 474150-474560 — Predicted thioestererase 1217 474885-476807 dinG Probable ATP-dependent helicase 1218 476820-477530 — Possible inactive homolog of metal-dependent 1219 proteases, putative molecular chaperone 477555 . . . 478106 — possible starvation-inducible outer membrane 1220 lipoprotein 478148-479836 lcfA Long chain fatty acid CoA ligase 1221 479892-481034 rnD Ribonuclease D 1222 C 481077-482945 — ✓ Conserved hypothetical acyltransferase 1223 C 483019-484110 — Predicted GTPase, probable translation factor 1224 C 484158-484742 Pth Peptidyl-tRNA hydrolase 1225 484925-485233 — Conserved hypothetical protein 1226 485236-486450 — Conserved hypothetical cupin superfamily 1227 metalloenzyme 486452-487771 xseA Exodeoxyribonuclease VII large subunit 1228 487948-488601 adpP ADP-ribose pyrophoaphatase 1229 488611-489435 Icc Predicted phosphohydrolase 1230 C 489533-489820 — Conserved hypothetical protein 1231 490017-491393 ompP1 Outer membrane protein P1 precursor 1232 491439-492008 Ogt Methylated-DNA-protein-cysteine 1233 492005-492676 mutH DNA mismatch repair protein MutH 1234 C 492684-493976 mesJ Putative cell cycle protein MesJ 1235 C 493976-494080 — Pseudogene for pyridoxamine kinase 1236 C 494153-495100 accA Acetyl-coenzyme A carboxylase carboxyl 1237 transferase subunit alpha C 495189-495974 znuB High affinity zinc uptake system membrane 1238 protein ZnuB C 495974-496780 znuC High-affinity zinc uptake system ATP binding 1239 protein AnuC 495652-498379 — Conserved hypothetical metalloprotease 1240 498494-499450 tyrR Transcriptional regulatory protein TyrR 1241 C 499546-499821 hfq host factor-I protein Hfq 1242 C 499912-500907 rluC ribosomal large subunit pseudouridine synthase C 1243 501253-504108 rne ribonuclease E 1244 504340-504552 — Conserved hypothetical protein 1245 504876-505673 thiM hydroxyethylthiazole kinase 1246 505666-506475 thiD phosphomethylpyrimidine kinase 1247 506486-507166 thiE thiamine-phosphate pyrophosphorylase 1248 507150-508454 — Conserved hypothetical metabolite transport 1249 C 508466-508567 — hypothetical protein 1250 508623-510002 — putative protease 1251 C 510036-511355 srmB ATP-dependent RNA helicase SrmB 1252 511401-512123 — predicted O-methyltransferase 1253 C 512154-512759 — pseudogene for conserved hypothetical 1254 tRNA/rRNA methyltransferase 512906-514273 pssA CDP-diacylglycerol--serine O- 1255 phosphatidyltransferase C 514319-515044 fadR fatty acid metabolism regulator protein 1256 515145-516713 nhaB Na(+)/H(+) antiporter 2 1257 516723-517256 dsbB disulfide bond formation protein B 1258 C 517311-519143 glmS glucosamine--fructose-6-phosphate 1259 aminotransferase C 519256-519666 hupA DNA-binding protein HU 1260 C 519668-520258 — conserved hypothetical protein 1261 C 520294-521088 nudC NADH pyrophosphatase 1262 C 521155-521751 orfG conserved hypothetical 21.9 KD protein in 1263 locus involved in transformation C 521827-522513 comF competence protein F 1264 C 522526-523863 comE competence protein E 1265 C 523873-524283 comD competence protein D 1266 C 524283-524804 comC competence protein C 1267 C 524801-525307 comB competence protein B 1268 C 525308-526105 comA competence protein A 1269 526204-528798 mrcA penicillin-binding protein 1A 1270 528871-529716 — conserved hypothetical protein 1271 529849-530214 — conserved hypothetical protein 1272 530345-530947 recR recombination protein RecR 1273 530963-532918 topB DNa topoisomerase III 1274 533027-533368 secG protein-export membrane protein SecG 1275 C 533913-535583 fruA PTS system, fructose-specific IIBC component 1276 C 535585-536526 fruK 1-phosphofructokinase 1277 C 536528-538027 fruB PTS system, fructose-specific IIA/FPr 1278 component C 538099-538635 — conserved hypothetical protein 1279 C 538722-539000 vapD virulence-associated protein D 1280 C 539009-539200 vapX VapX 1281 C 539272-540570 — conserved hypothetical protein 1282 C 540621-541145 — conserved hypothetical protein 1283 C 541172-541954 — putative deoxyribonuclase 1284 C 542009-542992 holB DNA polymerase III, delta subunit 1285 C 542989-543117 — ✓ hypothetical protein 1286 543192-545315 lav ✓ autotransported protein Lav 1287 C 546221-546880 tmk thymidylate kinase 1288 C 546870-547913 — predicted periplasmic solute-binding protein 1289 C 547983-548924 surA survival protein SurA homolog 1290 C 548994-549533 pyrR PyrR bifunctional protein 1291 549665-550456 mazG predicted pyrophosphatase MazG 1292 C 550506-551381 — conserved hypothetical protein 1293 551574-553985 lon ATP-dependent protease La 1294 554102-555253 — predicted Fe—S oxidoreductase 1295 555387-556046 rpiA ribose 5-phosphate isomerase A 1296 556076-557308 serA D-3-phosphoglycerate dehydrogenase 1297 C 557351-558193 — predicted aminomethyltransferase related to 1298 GcvT C 558203-559066 — conserved hypothetical stress-induced protein 1299 559515-560426 hisG ATP phosphoribosyltransferase 1300 560519-561802 hisD histidinol dehydrogenase 1301 561871-562974 hisC hisitidinol-phosphate aminotransferase 1 1302 563086-564174 hisB histidine biosynthesis bifunctional protein HisB 1303 564240-564839 hisH imidazole glycerol phosphate synthase subunit 1304 HisH 564875-565624 hisA 1-5-[methylideneamino] imidazole-4- 1305 carboxamine isomerase 565606-566382 hisF imidazole glycerol phosphate synthase subunit 1306 HisF 566382-567047 hisI histidine biosynthesis bifunctional protein hisIE 1307 567129-567281 — conserved hypothetical protein 1308 567311-568513 tyrP tyrosine-specific transportprotein 1 1309 C 568699-569127 atpC ATP synthase epsilon chain 1310 C 569157-570530 atpD ATP synthase beta chain 1311 C 570547-571416 atpG ATP synthase gamma chain 1312 C 571432-572973 atpA ATP synthase alpha chain 1313 C 572986-573519 atpH ATP synthase delta chain 1314 C 573532-574002 atpF ATP synthase B chain 1315 C 574052-574306 atpE ATP cynthase C chain 1316 C 574362-575150 atpB ATP synthase A chain 1317 C 575187-575561 — predicted F0F1-type ATP synthase subunit I 1318 C 575687-576298 gidB methyltransferase GidB 1319 C 576438-576815 — conserved hypothetical protein 1320 576931-577533 — predicted phosphatase/phosphohexomutase 1321 577533-578006 — predicted membrane protein 1322 578191-578694 luxS S-ribosylhomocysteinase 1323 C 579638-580345 aphA Class B acid phosphatase 1324 580565-581092 hslV ATP-dependent protease HslV 1325 581103-582437 hslU HslU, ATP-dependent chaperone of the HslUV 1326 protease C 582507-583559 ptoD2 spermidine/putrescine-binding periplasmic 1327 protein 2 precursor 583699-584664 ordL probable oxidoereductase OrdL 1328 585158-586600 rmuC DNA recombination protein RmuC 1329 586764-587183 rbsD ribose transport permease protein 1330 587197-588678 rbsA ribose transport ATP-binding protein 1331 588691-589662 rbsC ribose transport permease protein 1332 589682-590560 rbsB ribose-binding periplasmic protein 1333 590658-591578 rbsK ribokinase 1334 591606-592604 rbsR ribose operon repressor 1335 592710-893261 — predicted membrane protein 1336 C 593401-593889 menG S-adenosylmethionine:2-demtyhylmenaquinone 1337 methyltransferase C 593491-594873 menA 1,4-dihydroxy-2-naphthoate 1338 octaprenyltransferase 594920-595639 — conserved hypothetical protein 1339 C 595692-596678 tehA tellurite resistance protein 1340 C 596830-601080 rpoC DNA-directed RNA polymerase beta′ chain 1341 C 601287-605318 rpoB DNA-directed RNA polymerase beta chain 1342 C 605702-606391 rplA 50S ribosomal protein L1 1343 C 606396-606824 rplK 50S ribosomal protein L11 1344 C 606997-607713 deoD purine nucleoside phosphorylase 1345 C 607797-609050 — predicted nucleoside permease 1346 C 609149-609937 — predicted pyruvate-formate lyase-activating 1347 enzyme C 609946-611490 — conserved hypothetical glycyl radical protein 1348 611703-612359 — predicted membrane protein 1349 C 612315-613385 waaQ ADP-heptose--lipooligosaccharide 1350 heptosyltransferase III C 613423-614502 fba frustose-biphosphate aldolase 1351 C 614613-615773 pgk phosphoglycerate kinase 1352 C 615869-616663 — probable rubonuclease I 1353 616741-617001 — putative ferredoxin-like protein 1354 C 617135-618355 tyrQ tyrosine-specific transport protein 2 1355 C 618392-618973 tdk thymidine kinase 1356 C 618982-620010 gcp probable O-sialoglycoprotein endopeptidase 1357 620242-620457 rpsU 30S ribosomal protein S21 1358 620591-622372 — DNA primase 1359 622443-624314 rpoD DNA polymerase sigma factor RpoD 1360 C 624624-626051 aspA aspartate ammonia-lyase 1361 C 626198-627013 ureH urease accessory protein UreH 1362 C 627088-627765 ureG urease accessory protein UreG 1363 C 627868-628575 ureF urease accessory protein UreF 1364 C 628560-629117 ureE urease accessory protein UreE 1365 C 629245-630963 ureC urease alpha subunit 1366 C 630975-631280 ureB urease beta subunit 1367 C 631357-631659 ureA urease gamma subunit 1368 631822-632112 groES 10 kDa chaperonin 1369 632135-633772 groEL 60 kDa chaperonin 1370 C 633946-634395 rplI 50S ribosomal protein L9 1371 C 634412-634639 rpsR 30S ribosomal protein S18 1372 C 634652-634798 priB primsomal replication protein N 1373 C 634965-635342 rpsF 30S ribosomal protein S6 1374 C 635534-635788 infA translation initiation factor IF-1 1375 635963-637037 lic2C ✓ pseudogene for UDP-glucose-- 1376 lipoligosaccharide glucosyltransferase 637121-637987 ksgA dimethyladenosine transferase 1377 C 638024-638962 lic2A UDP-Gal--lipooligosaccharide 1378 galactosyltransferase 639015-639842 apaH bis-tetraphosphatase, symmetrical 1379 639852-640475 — conserved hypothetical protein 1380 C 640601-642055 gnd 6-phosphogluconate dehydrogenase 1381 decarboxylating C 642171-642713 — conserved hypothetical protein 1382 C 642796-642984 — conserved hypothetical protein 1383 C 643054-643791 devB 6-phosphgluconolactonase 1384 C 643895-645379 zwf glucose-6-phosphate 1-dehydrogenase 1385 C 645455-646264 cysQ cysQ 1386 C 646269-646685 — conserved hypothetical protein 1387 C 646742-648724 — predicted membrane protein 1388 648953-649348 hslR heat shock protein 15 homolog 1389 C 649374-649826 asnC regulatory protein AsnC 1390 649980-650972 asnA aspartate--ammonia ligase 1391 651035-651388 — ✓ conserved hypothetical transposase-like protein 1392 652001-652858 — ✓ hypothetical protein 1393 C 653015-653131 — ✓ hypothetical protein 1394 653423-653563 — ✓ hypothetical protein 1395 C 653743-654417 gph phosphoglycolate phosphatase 1396 C 654463-655167 rpe ribulose-phosphate 3-epimerase 1397 C 655289-657709 gyrB DNA gyrase subunit B 1398 C 657835-660147 — predicted transcriptional accessory protein 1399 660243-660719 greB transcription elongation factor GreB 1400 C 660789-661406 — conserved hypothetical transcriptional regulator 1401 C 661406-662311 oxyR hydrogen peroxide-inducible genes activator 1402 662434-663159 pdgC peroxiredoxin•glutaredoxin 1403 C 663251-663472 slyX slyX 1404 663570-664295 fkby probably FKBP-type peptidyl-proyl cis-trans 1405 isomerase 664385-665050 — conserved hypothetical protein 1406 665050-665430 — uncharacterized conserved protein involved in 1407 intracellular sulfur reduction 665427-665786 — uncharacterized conserved protein involved in 1408 oxidation of intracellular sulfur 665795-666082 — uncharacterized conserved protein involved in 1409 oxidation of intracellular sulfur C 666213-667397 tufB elongation factor Tu 1410 668103-669026 coaA pantothenate kinase 1411 C 669117-670064 rseB sigma-E factor regulatory protein RseB 1412 C 670144-670719 rseA sigma-E factor negative regulator protein 1413 homolog C 670744-671313 rpoE RNA polymerase sigma-E factor 1414 C 671422-671679 — conserved hypothetical protein 1415 C 671769-672155 mscL large-conductance mechanosensitive channel 1416 C 672228-673604 trkA Trk system potassium uptake protein TrkA 1417 C 673617-674969 sun SUN protein 1418 C 674969-675925 fmt methionyl-tRNA formyltransferase 1419 C 676026-676535 def peptide deformylase 1420 676725-677162 — ✓ hypothetical protein 1421 677140 . . . 677325 — ✓ hypothetical protein 1422 C 683606-684259 sxy DNA transformation protein TfoX 1423 684631-685695 recA RecA 1424 685774-686232 recX regulatory protein RecX 1425 C 686229-686615 crcB CrcB 1426 C 686615-687433 — predicted hydrolase of the HAD superfamily 1427 687836-688840 argF ornithine carbamoyltransferase, catabolic 1428 688850-689782 arcC carbamate kinase 1429 689887-691416 — predicted membrane protein 1430 C 691826-694980 hgpD ✓ pseudogene for hemoglobin-haptoglobin 1431 binding protein D 696052-696756 pepE peptidase E 1432 696774-698069 — predicted C4-dicarboxylate transporter 1433 698141-699412 abgA aminobenzoyl-glutamate utilization protein A 1434 699505-701478 cpdB 2′,3′-cyclic-nucleotide 2′-phosphodiesterase 1435 C 701587-701994 — ✓ HTH-type transcriptional regulator 1436 C 702004-702681 — ✓ zinc transported ZitB 1437 703189-705078 gidA glucose inhibited division protein A 1438 705260-705634 rpsL 30S ribosoomal protein S12 1439 705791-706261 rpsG 30S ribosomal protein S7 1440 706345-708447 fusA elongation factor G 1441 708512-709696 tufB2 elongation factor Tu 1442 710156-710500 — ✓ predicted chloride channel protein 1443 710491-710673 — predicted chloride channel protein 1444 710676-711659 dusA tRNA-dihydrouridine synthase A 1445 C 711707-711997 — conserved hypothetical protein 1446 C 712020-713030 trpS tryptophanyl-tRNA synthetase 1447 713153-713770 — predicted protein involved in purine metabolism 1448 713793-715163 purB adenylosuccinate lyase 1449 715435-715926 rplJ 50S ribosomal protein L10 1450 715981-716532 rplL 50S ribosomal protein L7/L12 1451 716511-717881 glmU bifunctional GlmU protein 1452 C 717916-718227 — hypothetical protein 1453 C 718766-719701 pldB probable lysophospholipase L2 1454 C 719755-720820 asd aspartate-semialdehyde dehydrogenase 1455 C 721036-721752 — conserved hypothetical protein 1456 721944-722600 — ✓ predicted 2-methylthioadenine synthetase 1457 C 722664-723242 mdaB putative NADPH-quinone reductase, modulator 1458 of drug activity B 723521-725533 rep ATP-dependent DNA helicase rep 1459 C 725539-725751 — predicted periplasmic lipoprotein 1460 C 725748-726218 kdtB phosphopantetheine adenylyltransferase 1461 C 726215-727498 kdtA 3-deoxy-D-manno-octulosonic acid transferase 1462 727561-728325 lgtF UDP-glucose--lipooligosaccharide 1463 glucosyltransferase C 728322-728885 tag DNA-3-methyladenine glycolase 1464 C 729000-729752 — ✓ hypothetical protein 1465 C 729764-730570 aroE shikimate 5-dehydrogenase 1466 C 730574-731125 predicted translation factor SUA5 1467 C 731141-731677 — Zn-finger domain associated with 1468 topoisomerase typr I C 731687-733603 — probable ABC transporter, ATP binding protein 1469 C 733788-734084 — putative HTH-type transcriptional regulator 1470 C 734077-734259 — ✓ conserved hypothetical protein 1471 C 734529-737507 hgpB ✓ hemoglobin-haptoglobin binding protein B 1472 738238-739983 — hypothetical ABC transporter, ATP-binding 1473 protein 739976-741634 — probable ABC transporter, ATP binding protein 1474 C 741718-742749 — conserved hypothetical protein 1475 C 742746-743066 — conserved hypothetical protein 1476 C 743054-743362 — putative HTH-type transcriptional regulator 1477 C 743567-744568 glpX fructose-1,6-bisphosphatase class II GlpX 1478 744730-744948 — conserved hypothetical protein 1479 745047-745487 mioC MioC 1480 745484-745918 dtd D-tyrosyl-tRNA (Tyr) deacylase 1481 C 746225-746701 ispF 3-C-methyl-D-erythritol 2,4-cyclodiphosphate 1482 synthase C 746698-747375 ispD 2-C-methyl-D-erythritol 4-phosphate 1483 cytidylyltransferase C 747375-747653 ftsB cell division protein FtcB 1484 C 747776-748243 gpt xanthine-guanine phosphoribosyltransferase 1485 748354-749808 pepD aminoacyl-histidine dipeptidase 1486 C 749861-750748 xerC site-specific recombinase XerC 1487 C 750742-751182 — conserved hypothetical protein 1488 751348-752138 tpiA triosephosphate isomerase 1489 C 752221-752538 glpE thiosulfate sulfurtransferase GlpE 1490 C 752539-753417 — conserved hypothetical protein 1491 C 753449-754327 ilvY HTH-type transcriptional activator IlvY 1492 755011-756489 ilvC ketol-acid reductoisomerase 1493 C 756600-757880 glpC anaerobic glycerol-3-phosphate dehydrogenase 1494 subunit C C 757891-759189 glpB anaerobic glycerol-3-phosphate dehydrogenase 1495 subunit B C 759179-760870 glpA anaerobic glycerol-3-phosphate dehydrogenase 1496 subunit A 761163-762605 glpT glycerol-3-phosphate transporter 1497 762709-763803 glpQ glycerophosphoryl diester phosphodiesterase 1498 precursor 764033-764827 glpF glycerol uptake facilitator protein 1499 764848-766359 glpK glycerol kinase 1500 C 766443-766910 gpt2 xanthine-guanine phosphoribosyltransferase 1501 766932-767069 — pseudogene for glycerophosphoryl diester 1502 phosphodiesterase precursor 767195-768019 hel outer membrane protein P4, NADP phosphatase 1503 768186-768869 rluE ribosomal large subunit pseudouridine synthase E 1504 C 768998-770002 ppx pseudogene for putative exopolyphosphatase 1505 C 769974-773858 — conserved hypothetical protein 1506 C 773868-775604 — conserved hypothetical protein 1507 C 775755-776327 slyD FKBP-type peptidyl-prolyl cis-trans isomerase 1508 SlyD C 776405-776827 — conserved hypothetical protein 1509 776936-777955 truD tRNA pseudouridine synthase D 1510 777965-778714 surE acid phosphatase surE 1511 778723-779301 — conserved hypothetical protein 1512 779310-779495 — conserved hypothetical protein 1513 779530-780747 lppB outer membrane antigenic lipoprotein B 1514 781115-782533 tnaA ✓ tryptophanase 1515 782617-783849 tnaB ✓ tryptophan-specific transport protein 1516 783990-786575 mutS DNA mismatch repair protein MutS 1517 786781-788166 selA L-seryl-tRNA selenium transferase 1518 788163-790022 selB selenocysteine-specific elongation factor 1519 790041-790895 — ✓ hypothetical protein 1520 791015-791311 — conserved hypothetical protein 1521 791311 . . . 791619 — conserved hypothetical protein 1522 C 791676-794868 hgpC ✓ pseudogene for hemoglobin-haptoglobin 1523 utilization protein C 795165-796463 tig trigger factor 1524 796586-797167 clpP ATP-dependent clp protease proteolytic subunit 1525 797177-798412 clpX ATP-dependent Clp protease ATP-binding 1526 subunit ClpX 798557-798973 secE preprotein translocase SecE 1527 798975-799532 nusG transcription antitermination protein NusG 1528 799683-800435 vacJ VacJ lipoprotein 1529 800489-800881 — putative translation initiation inhibitor YjgF 1530 family 801049-801900 htpX probable protease HtpX 1531 C 801942-802223 sirA SirA 1532 802297-802917 — conserved hypothetical protein 1533 802290-804383 trkH Trk system potassium uptake protein TrkH 1534 810818-811237 psiE PsiE 1535 C 811301-812587 hemY HemY 1536 C 812598-813809 hemX putative uroporphyrin-III C-methyltransferase 1537 814115-816646 cya adenylate cyclase 1538 816720-717727 gpsA glycerol-3-phosphate dehydrogenase 1539 817744-818547 cysE serine acetyltransferase 1540 818557-819372 — conserved hypothetical shikimate 5- 1541 dehydrogenase-like protein 819510-820895 — possible di- and tricarboxylate transporter 1542 C 821033-821881 folD FolD bifunctional protein 1543 C 822409-823695 fucP L-fucose permease 1544 C 823734-824384 fucA L-fuculose phosphate aldolase 1545 C 824404-824838 fucU fucose operon protein FucU 1546 C 824852-826264 fucK L-fuculokinase 1547 C 826337-828151 fucI L-fucose isomerase 1548 C 828336-829085 fucR L-fucose operon activator 1549 829284-832055 hepA RNA polymerase associated protein homolog 1550 832058-832717 rluA ribosomal large subunit pseudouridine synthase A 1551 832744-833322 glpG GlpG 1552 833352-834119 glpR glycerol-3-phosphate regulon repressor 1553 C 834421-835242 metQ probable D-methionine-binding lipoprotein 1554 MetQ C 835281-835970 metI probable D-methionine transport system 1555 permease protein C 835960-836997 metN probable D-methionine transport ATP-binding 1556 protein 837173-837727 — conserved hypothetical protein 1557 843979-844539 — conserved hypothetical protein 1558 844543-844983 — conserved hypothetical protein 1559 C 845045-845671 narP nitrate/nitrite response regulator protein 1560 C 845877-847124 lysA diaminopimelate decarboxylase 1561 C 847172-847303 — hypothetical protein 1562 847386-847691 cyaY CyaY 1563 847693-849552 recQ ATP-dependent DNA helicase RecQ 1564 849637-851355 proS prolyl-tRNA synthetase 1565 C 851443-853791 ostA organic solvent tolerance protein 1566 C 853914-855323 sufI SufI 1567 C 855325-856047 plsC 1-acyl-sn-glycerol-3-phosphate acyltransferase 1568 856133-856846 lpxH UDP-2,3-diacylglucosamine hydrolase 1569 C 857076-858602 — conserved hypothetical sodium dependent 1570 transporter 858849-859475 ilvG acetolactate synthase isozyme II large subunit 1571 859517-861355 ilvD dihydroxy-acid dehydratase 1572 861435-862976 thdI threonine dehydratase biosynthetic 1573 863014-866493 dnaE DNA polymerase III alpha subunit 1574 866732-868375 pgmB phosphoglucomutase 1575 C 868542-869051 secB protein-export protein SecB 1576 C 869065-869511 — predicted rhodanese-related sulfurtransferase 1577 869759-871081 dcuB anaerobic C4-dicarboxylate transporter DcuB 1578 871285-872619 ndhA NADH dehydrogenase 1579 C 872699-875131 plsB glycerol-3-phosphate acyltransferase 1580 875380-876006 lexA LexA repressor 1581 876152-876976 dapF diaminopimelate epimerase 1582 877196-877693 tpx probable thiol peroxidase 1583 878256-882218 purL phosphoribosylformylglycinamidine synthase 1584 882414-882652 lex2A ✓ pseudogene for Lex2A homolog 1585 882724-883467 lex2B ✓ UDP-glucose--lipooligosaccharide 1586 glucosyltransferase C 883528-884370 — conserved hypothetical protein 1587 C 884370-885602 — predicted membrane-bound metallopeptidase 1588 885778-886461 gpmA 2,3-bisphosphoglycerate-dependent 1589 phsphoglycerate mutase C 886539-886751 rpL31 50S ribosomal protein L31 1590 886928-888064 mutY A/G-specific adenine glycosylase 1591 888042-888314 — conserved hypothetical protein 1592 888329-889402 mltC membrane-bound lytic murein transglycolase C 1593 precursor C 890013-890693 — predicted diadenosine tetraphosphatase and 1594 related serine/threonine protein phosphatase C 890690-891955 nadR bifunctional protein NadR 1595 892174-892821 ribB 3,4-dihydroxy-2-butanone 4-phosphate 1596 synthase 892836-892684 lpsA lipooligosaccharide glycosyl transferase 1597 C 893733-894215 — conserved hypothetical tRNA/rRNA 1598 methyltransferase C 894225-894806 — predicted N6-adenine-specific methylase 1599 894861-896105 FtsY cell division protein FtsY 1600 896124-896780 ftsE cell division ATP-binding protein EtsE 1601 896790-897722 ftsx cell division protein ftsx 1602 C 897817-898998 atoB acetyl-CoA acetyltransferase 1603 C 899013-900356 atoE short chain fatty acids transporter 1604 C 900359-901024 atoA acetate CoA-transferase beta subunit 1605 C 901035-901688 atoD acetate CoA-transferase alpha subunit 1606 901894-902841 — putative HTH-type transcriptional regulator 1607 903078-903389 rpsJ 30S ribosomal protein S10 1608 903406-904032 rplC 50S ribosomal protein L3 1609 904048-904650 rplD 50S ribosomal protein L4 1610 904647-904946 rplW 50S ribosomal protein L23 1611 904964-905785 rplB 50 S ribosomal protein L2 1612 905811-906086 rpsS 30S ribosomal protein S19 1613 906098-906430 rplV 50S ribosomal protein L22 1614 906448-907155 rpsC 30S ribosomal protein S3 1615 607169-907579 rplP 50S ribosomal protein L16 1616 907579-907770 rpmC 50S ribosomal protein L29 1617 907770-908027 rpsQ 30S ribosomal protein S17 1618 908417-909040 — conserved hypothetical protein 1619 909321-909692 rplN 50S ribosomal protein L14 1620 909703-910014 rplX 50S ribosomal protein L24 1621 910032-910571 rplE 50S ribosomal protein L5 1622 910583-910888 rpsN 30S ribosomal protein S14 1623 910925-911317 rpsH 30S ribosomal protein S8 1624 911333-911866 rplF 50S ribosomal protein L6 1625 911880-912233 rplR 50S ribosomal protein L18 1626 912248-912748 rpsE 30S ribosomal protein S5 1627 912755-912934 rpmD 50S ribosomal protein L30 1628 912938-913372 rplO 50S ribosomal protein L15 1629 913380-914705 secY preprotein translocase SecY subunit 1630 914987-915343 rpsM 30S ribosomal protein S13 1631 915359-915748 rpsK 30S ribosomal protein S11 1632 915776-916396 rpsD 30S ribosomal protein S4 1633 916428-917414 rpoA DNA-directed RNA polymerase alpha chain 1634 917455-917841 rplQ 50S ribosomal protein L17 1635 C 917968-918564 — predicted cAMP-binding protein—catabolite 1636 gene activator and regulatory subunit of cAMP- dependent protein kinase C 918683-919383 — pseudogene for conserved hypothetical protein, 1637 predicted arylsulfatase regulator, Fe—S oxidoreductase 919587-920326 — predicted permease 1638 C 920358-921551 dxr 1-deoxy-D-xylulose 5′phosphate 1639 reductoisomerase C 921661-922218 frr ribosome recycling factor 1640 C 922370-923986 pckA phosphoenolpyruvate carboxykinase 1641 C 924159-925040 hslO 33 kDa chaperonin 1642 C 925185-926558 argH argininosuccinate lyase 1643 C 926693-927580 galU UTP-glucose-1-phosphate uridylyltransferase 1644 C 927602-927793 csrA carbon storage regulator homolog 1645 C 927871-930495 alaS alanyl-tRNA synthetase 1646 C 930682-931107 uspA universal stress protein A 1647 C 931209-932501 pepP Xaa-Pro aminopeptidase 1648 C 932513-933061 — conserved hypothetical protein 1649 C 933232-934257 galM aldose 1-epimerase 1650 C 934264-935460 galK glactokinase 1651 C 935495-936544 galT galactose-1-phosphate uridylyltransferase 1652 936752-937750 galR HTH-type transcriptional regulator GalR 1653 937841-938890 mglB D-galactose-binding periplasmic protein 1654 precursos 938956-940476 mglA galactoside transport ATP-binding protein 1655 MglA 940493-941503 mglC galactoside transport system permease protein 1656 MglC 941649-942383 — conserved hypothetical protein 1657 942389-942946 ispZ probable intracellular septation protein A 1658 942946-943410 — putative acyl-CoA thioester hydrolase 1659 943427-943723 — conserved hypothetical protein 1660 943739-945520 slt putative soluble lytic murein transglycosylase 1661 945558-945863 trpR Trp operon repressor 1662 946006-946590 mtgA monofuctional biosynthetic peptidoglycan 1663 transglycolase C 946696-947040 frdD fumarate reductase subunit D 1664 C 947053-947463 frdC fumarate reductase subunit C 1665 C 947474-948244 frdB fumarate reductase iron-sulfur protein 1666 C 948327-950036 frdA fumarate reductase flavoprotein subunit 1667 950217-951218 genX putative lysyl-tRNA synthetase 1668 C 951484-952167 cpxR transcriptional regulatory protein CpxR 1669 C 952221-952634 smpA small protein A 1670 C 952700-953725 ndpA nucleosid-associated protein NdpA 1671 953842-954060 — conserved hypothetical protein 1672 954062-955819 — predicted hydrolase of alkaline phosphatase 1673 superfamily 956017-957009 — conserved hypothetical protein 1674 C 957148-957726 mobA probable molybdopterin-guanine dinucleotide 1675 biosyntheses protein A 957791-958057 — conserved hypothetical protein 1676 958068-958685 dsbA thiol:disulfide interchange protein DsbA 1677 958747-959085 — conserved hypothetical protein 1678 959226-960317 trmA tRNA (Uracil-5)-methyltransferase 1679 960506-961072 — conserved hypothetical protein 1680 961066-961500 — predicted positive regulator of Sigma E 1681 961901-962437 mobB molybdopterin-guanine dinucleotide 1682 biosynthesis protein B 962434-963825 — conserved hypothetical protein 1683 963966-965615 hbpA heme-binding protein A 1684 965791-966552 — putative heme iron utilization protein 1685 C 966689-967036 — conserved hypothetical protein 1686 C 967051-969843 polA DNA polymerase I 1687 969992-970294 — conserved hypothetical protein 1688 970617-971180 — predicted 5-formyltetrahydrofolate cyclo-ligase 1689 971308-973878 clpB ClpB 1690 C 973916-974656 — probable tRNA/rRNA methyltransferase 1691 C 974712-977060 vacB ribonuclease R 1692 C 977252-977959 — conserved hypothetical protein 1693 978095-978784 pdxH pyridoxamine 5′-phosphate oxidase 1694 C 978871-980721 typA GTP-binding protein TypA/BipA 1695 C 980848-981828 lic3A2 CMP-neu5Ac--lipooligosaccharide alpha 2-3 1696 sialyltransferase 981985-983397 glnA glutamine synthetase 1697 983484-984527 rmlB ✓ dTDP-glucose 4,6-dehydratase 1698 984604-985908 pepB peptidase B 1699 985915-986340 ndk nucleoside diphosphate kinase 1700 C 986410-987582 — conserved hypothetical GTP-binding protein 1701 C 987610-988530 — conserved hypothetical transport protein 1702 C 988610-988867 rpmA 50S ribosomal protein L27 1703 C 988888-989199 rplU 50S ribosomal protein L21 1704 989412-990401 ispB octaprenyl-diphosphate synthase 1705 990452-991189 — conserved hypothetical protein 1706 C 991249-992619 — predicted Na+/alanine symporter 1707 C 993019-993729 arcA aerobic respiration control protein ArcA 1708 993921-995660 dsbD thiol:disulfide interchange protein DsbD 1709 995777-996181 — predicted membrane protein 1710 996370-997968 purH bifunctional purine biosynthesis protein PurH 1711 998064-999353 purD phosphoribosylamine--glycine ligase 1712  999509-1001076 — ✓ pseudogene for predicted membrane-associated, 1713 metal-dependent hydrolase 1001199-1002464 glyA serine hydroxymethyltransferase 1714 1002523-1003155 coaE dephospho-CoA kinase 1715 1003148-1003354 — conserved hypothetical zinc-binding protein 1716 1003463-1004719 rh1B ATP-dependent RNA helicase Rh1B 1717 1004961-1005524 — hypothetical transcriptional regulator 1718 1005556-1006752 — predicted membrane-fusion protein 1719 1006752-1009850 — predicted cation/multidrug efflux pump 1720 1010385-1010855 — predicted cell division protein 1721 C 1010868-1012400 emrB multidrug resistance protein 1722 C 1012410-1013582 emrA multidrug resistance protein A 1723 C 1013755-1014237 folA dihydrofolate reductase 1724 1014339-1015445 proB glutamate 5-kinase 1725 1015526-1016116 nudH probable nucleoside polyphosphate hydrolase 1726 1016116-1016910 — predicted permease 1727 1016919-1017725 lgt prolipoprotein diacylglyceryl transferase 1728 1017735-1018586 thyA thymidylate synthase 1729 1018586-1019107 — conserved hypothetical protein 1730 C 1019135-1019449 — conserved hypothetical protein 1731 1019520-1019831 — conserved hypothetical protein 1732 1019956-1022661 secA preprotein translocase SecA subunit 1733 1022726-1023136 mutT mutator protein MutT 1734 1023245-1025101 kefB glutathione-regulated potassium-efflux system 1735 protein 1025202-1025966 — conserved hypothetical SAM-dependent 1736 methtransferase 1026111-1026866 rpsB 30S ribosomal protein S2 1737 1027000-1027851 tsf elongation factor Ts 1738 C 1028031-1029056 lpxD UDP-3-O-[3-hydroxymyristoyl] glucosamine 1739 N-acyltransferase C 1029069-1029662 omp26 outer membrane protein 26 1740 C 1029770-1032172 — protective surface antigen D15 1741 C 1032192-1033523 — predicted membrane bound zinc 1742 matalloprotease with PDZ domain C 1033533-1034399 cdsA phosphaatidate cytidylyltransferase 1743 C 1034417-1035136 uppS undecaprenyl pyrophosphate synthetase 1744 1035385-1037970 leuS leucyl-tRNA synthetase 1745 1038020-1038568 — conserved predicted lipoprotein 1746 1038568-1039602 holA DNA polymerase III, delta subunit 1747 C 1039836-1042004 glyS glycyl-tRNA synthetase beta chain 1748 C 1042050-1042466 — ✓ hypothetical protein 1749 C 1042606-1042971 — conserved hypothetical protein 1750 C 1043001-1043261 — glutaredoxin-related protein 1751 C 1043320-1044228 glyQ glycyl-tRNA synthetase alpha chain 1752 1044491-1046017 hktE catalase 1753 C 1046249-1047430 — predicted glutathionylspermidine synthase 1754 C 1047431-1048012 — conserved hypothetical protein 1755 C 1048025-1048477 — conserved hypothetical protein 1756 C 1048528-1049838 eno enolase 1757 C 1049959-1051164 — conserved hypothetical protein 1758 C 1051161-1052303 nrfF formate-dependent nitrite reductase complex 1759 nrfFG subunit C 1052300-1052830 dsbE2 probable thiol:disulfide interchange protein 1760 DsbE C 1052830-1054737 nrfE cytochrome c-type biogenesis protein NrfE 1761 C 1054846-1055658 suhB inositol-1-monophosphatase 1762 1055858-1056370 — conserved hypothetical protein 1763 1056370-1057089 — predicted Type II secretory pathway, PulJ-like 1764 protein 1057086-1057769 — conserved hypothetical protein 1765 1057774-1058049 — conserved hypothetical protein 1766 1058095-1061460 — exodeoxyribonuclease V gamma chain 1767 1061515-1061964 — predicted transcriptional regulator 1768 1061967-1063085 ribD riboflavin biosynthesis protein RibD 1769 11063086-1064108 degS protease DegS 1770 C 1064182-1064997 mutM formamidopyrimidine-DNA glycosylase 1771 C 1065229-1066764 ddc L-2,4-diaminobutyrate decarboxylase 1772 C 1066784-1067182 — predicted nucleic acid-binding protein, contains 1773 PIN domain C 1067182-1067415 — conserved hypothetical protein 1774 C 1067562-1068926 dat diaminobutyrate--2-oxoglutarate 1775 aminotransferase C 1069273-1069443 rpmG 50S ribosomal protein L33 1776 C 1069455-1069691 rpmB 50S ribosomal protein L28 1777 C 1069905-1070609 radC DNA repair protein Radc homolog 1778 1070733-1071935 dfp phosphopantothenoylcysteine 1779 synthetase/decarboxylase 1071983-1072438 dut deoxyuridine 5′triphosphate 1780 nucleotidohydrolase 1072442-1073098 ttk ttk 1781 1073120-1073371 — hypothetical protein 1782 1073356-1074030 crp catabolite gene activator 1783 C 1074395-1075573 — hypothetical RNA methyltransferase 1784 C 1075566-1076621 nagZ beta-hexosaminidase 1785 C 1076625-1076975 — predicted periplasmic lipoprotein 1786 C 1076975-1077367 — HIT-like protein 1787 C 1077442-1080267 ileS isoleucyl-tRNA synthetase 1788 C 1080296-1081234 ribF riboflavin biosynthesis protein RibF 1789 C 1081269-1082801 mviN putative virulence factor MviN 1790 1083104-1083373 rpsT 30S ribosomal protein S20 1791 C 1083445-1084011 — conserved hypothetical protein 1792 1084157-1085014 menB naphthoate synthase 1793 1085184-1086173 menC O-succinylbenzoate synthase 1794 1086230-1086679 aroQ 3-dehydroquinate dehydratase 1795 1086833-1087300 accB biotin carboxyl carrier protein of acetyl-CoA 1796 carboxylase 1087477-1088823 accC biotin carboxylase 1797 1089028-1089285 — conserved hypothetical membrane protein 1798 1089282-1090736 panF sodium/pantothenase symporter 1799 1090857-1091729 — conserved hypothetical protein 1800 1091764-1092651 prmA ribosomal protein L11 methyltransferase 1801 1092798-1093778 dusB tRNA-dihydrouridine synthase B 1802 1093772-1094071 fis DNA-binding protein fis 1803 C 1094172-1094657 smpB SsrA-binding protein 1804 C 1094896-1095861 pfkA 6-phosphfructokinase 1805 C 1095957-1096538 — conserved hypothetical protein 1806 C 1096531-1097382 — conserved hypothetical protein 1807 C 1097360-1098481 smf smf 1808 1099025-1100572 leuA 2-isopropylmalate synthase 1809 1100735-1101811 leuB 3-isopropylmalate dehydrogenase 1810 1101988-1103397 leuC 3-isopropylmalate dehydratase large subunit 1811 1103422-1104024 leuD 3-isopropylmalate dehydratase small subunit 1812 C 1104138-1109522 Iga1 ✓ IgA-specific serine endopeptidase 1813 C 1109749-1110828 recF DNA replication and repair protein RecF 1814 C 1110830-1111930 dnaN DNA polymerase III, beta chain 1815 C 1112123-1113487 dnaA chromosomal replication initiator protein DnaA 1816 C 1113674-1116412 tbp1 transferrin-binding protein 1 1817 C 1116457-1118349 tbp2 transferrin-binding protein 2 1818 1118446-1119906 — conserved hypothetical protein 1819 1120082-1120216 rpmH 50S ribosomal protain L34 1820 1120229-1120588 rnpA ribonuclease P protein component 1821 1120552-1120812 — conserved hypothetical protein 1822 1120812-1122437 yidC proprotein translocase subunit YidC 1823 1123308-1124666 trmE probable tRNA moficiation GTPase TrmE 1824 1124855-1126723 ppiD peptidyl-prolyl cis-trans isomerase D 1825 1126811-1128370 — predicted PR--lipooligosaccharide 1826 phosphorylethanolamine transferase 1128440-1128955 lspA lipoprotein signal peptidase 1827 1128952-1129896 ispH 4-hydroxy-3-methylbut-2-enyl diphosphate 1828 reductase 1130065-1130412 — conserved hypothetical protein 1829 1130659-1131657 tbpA thiamine-binding periplasmic protein 1830 1131662-1133278 thiP thiamine transport system permease protein 1831 1133256-1133909 thiQ thiamine transport ATP-binding protein 1832 1134022-1135023 bioB biotin synthase 1833 C 1135148-1137145 tktA transketolase 1834 1137526-1138470 serB phosphoserine phosphatase 1835 1138488-1138979 — conserved hypothetical protein 1836 1139275-1140222 corA magnesium and cobalt transport protein CorA 1837 1140213-1140767 — predicted integral membrane protein 1838 C 1140947-1141831 — predicted glutamine amidotransferase 1839 C 1141988-1142515 — ✓ hypothetical protein 1840 1142645-1143847 — predicted ATPase 1841 C 1144037-1145386 — ✓ hypothetical protein 1842 C 1145854-1146354 — predicted ferredoxin 1843 C 1146354-1146964 — conserved hypothetical protein 1844 C 1147077-1147916 dmsC anaerobic dimethyl sulfoxide reductase chain C 1845 C 1147918-1148535 dmsB anaerobic dimenthyl sulfoxide reductase chain B 1846 C 1148546-1150966 dmaA anaerobic dimethyl sulfoxide reductase chain A 1847 1151219-1152328 — conserved hypothetical protein 1848 1152372-1152665 — putative mercuric transport MerT homolog 1849 1152674-1152952 — predicted copper chaperone MerP homolog 1850 C 1153077-1154921 — conserved hypothetical ABC transporter 1851 C 1155000-1155896 — conserved hypothetical transcriptional regulator 1852 1156008-1156349 — conserved putative gamma- 1853 carboxymuconolactone decarboxylase subunit C 1156460-1157056 — conserved hypothetical protein 1854 C 1157062-1159227 res putative type III restriction-modification sustem 1855 HindVIP enzyme res C 1159217-1161318 mod ✓ pseudogene for putative type III restriction- 1856 modification system HibdVIP enzyme mod C 1161534-1162127 rnhB ribonuclease HII 1857 C 1162120-1163292 lpxB lipid-A-disaccharide synthase 1858 C 1163359-1164147 lpxA acyl0[acyl-carrier-protein]--UDP-N- 1859 acetylglucosamine O-Acyltransferase C 1164161-1164607 fabZ (3R)-hydroxymyristoyl-[acyl carrier protein] 1860 dehydratase C 1164683-1166290 — predicted PR--lipooligosaccharide 1861 phosphorylethanolamine transferase 1166383-1167096 pyrH uridylate kinase 1862 C 1167335-1168300 nrfD NrfD, formate-dependent nitrite reductase, 1863 membrane component C 1168297-1168974 nrfC NrfC, Fe—S-cluster-containing hydrogenase 1864 component 1 1168971-1169654 nrfB NrfB, cytochrome C-type protein 1865 C 1169698-1171314 nrfA cytochrome c552 1866 C 1171476-1175384 hrpA ATP-dependent helicase HrpA homolog 1867 C 1175381-1175755 — conserved putative small membrane protein 1868 C 1175756-1176208 — conserved putative membrane protein 1869 C 1176474-1177610 cyoB probable cyrochrome oxidase subunit II 1870 C 1177625-1179190 cyoA probable cytochrome oxidase dubunit I 1871 1179843-1181480 pyrG CTP synthase 1872 C 1181625-1182305 pnuC nictinamide riboside transporter 1873 C 1182487-1183260 — probable amino-acid ABC transporter ATP- 1874 binding protein C 1183264-1183941 — probable amino-acid ABC transporter permease 1875 protein C 1183964-1184737 — probable amino-acid ABC transporter binding 1876 protein C 1185038-1186312 murA UDP-N-acetylglucosamine a- 1877 carboxyvinyltransferase C 1186323-1186580 — predicted transcriptional regulator, BolA 1878 superfamily C 1186601-1186918 — predicted NTP binding protein, contains STAS 1879 domain C 1186929-1187573 — conserved ABC-type transport system protein 1880 C 1187606-1188106 — conserved ABC-type transport system protein, 1881 periplasmic component C 1188116-1188901 — conserved ABC-type transport system protein, 1882 permease component C 1188898-1189692 — conserved ABC-type transport system protein, 1883 ATPase component C 1190098-1190745 sodA superoxide dismutase [Mn] 1884 1190994-1191632 ccmA heme exporter protein A 1885 1191637-1192302 ccmB heme exporter protein B 1886 1192363-1193103 ccmC heme exporter protein C 1887 1193146-1193349 ccmD heme exporter protein D 1888 1193346-1193867 ccmE cytochrome c-type biogenesis protein CcmE 1889 1193864-1195813 ccmF cytochrome c-type biogenesis protein CcmF 1890 1195953-1196498 dsbE thiol:disulfide interchange protein DsbE 1891 1196498-1197877 ccmH pseudogene for cytochrome c-type biogenesis 1892 protein CcmH precursor 1197998-1198489 — hypothetical protein 1893 1198491-1198799 — conserved hypothetical protein 1894 C 1198804-1200843 ligN DNA ligase 1895 C 1200928-1201914 zipA cell division protein ZipA 1896 1202067-1202885 cysZ CysZ 1897 1202894-1203934 cysK cysteine synthase 1898 1204182-1205406 — pseudogene for conserved hypothetical 1899 metabolite transport protein C 1205515-1206555 rfaF ADP-heptose--lipooligosaccharide 1900 heptosyltransferase II C 1206631-1207794 xylR xylose operon refulatory protein 1901 C 1207906-1209312 — conserved hypothetical Na(+)/H(+) antiporter 1902 C 1209314-1210486 aspC2 putative aspartate aminotransferase 1903 1210699-1212018 xylA xylose isomerase 1904 1212021-1213556 xylB xylulose kinase 1905 C 1213607-1214533 rfaD ADP-L-glycero-D-manno-heptose-6-epimerase 1906 C 1214586-1215089 — thioredoxin-like protein 1907 1215200-1215871 deoC deoxyribose-phosphate aldolase 1908 C 1215905-1217434 comM competence protein ComM 1909 C 1217549-1218166 engB Probable GTP-binding protein EngB 1910 1218259-1219137 — D-xylose transport permease protein 1911 C 1219177-1220175 oppF oligopeptide transport ATP-binsing protein 1912 C 1220172-1221143 oppD oligopeptide transport ATP-binding protein 1913 C 1221153-1222088 oppC oligopeptide transport system permease protein 1914 C 1222098-1223018 oppB oligopeptide transport system permease protein 1915 C 1223099-1224724 oppA periplasmic oligopeptide-binding protein 1916 1225019-1225972 talB Transaldolase 1917 1227033-1228634 — carbon starvation protein, predicted membrane 1918 protein 1228836-1229291 mraZ MraZ 1919 1229322-1230287 mraW predicted S-adenosylmethionine-dependent 1920 methyltransferase involved in cell envelope biogenesis 1230290-1230613 ftsL cell division protein FtsL 1921 1230626-1232458 ftsI peptidoglycan synthetase FtsI 1922 1232468-1233934 murE UDP-N-acetylmuramoylalanyl-D-glutamate-- 1923 2,6-diaminopimelate ligase 1233948-1235321 murF UDP-N-acetylmuramoyl-tripeptide--D-alanyl- 1924 D-alanine ligase 1235315-1236397 mraY phospho-N-acetylmuramoyl-pentapeptide- 1925 transferase 1236509-1237822 murD UDP-N-acetylmuramoylaalanine--D-glutamate 1926 ligase 1237845-1239029 ftsW cell division protein FtsW 1927 1239041-1240096 murG UDP-N-acetylglucosamine--N- 1928 acetylmuramylpyrophosphoryl-undecaprenol N-acetylglucosamine transferase 1240234-1241661 murC UDP-N-acetylmuramate--L-alanine ligase 1929 1241730-1242650 ddlB D-alanine--D-alanine ligase 1930 1242650-1243414 ftsQ cell division protein FtsQ 1931 1243433-1244710 ftsA cell division protein FtsA 1932 1244794-1246059 ftsZ cell division protein FtsZ 1933 1246098-1247015 lpxC UDP-3-O-[3-hydroxymyristoyl] N- 1934 acetylglucosamine deacetylase 1247142-1248299 pheA P-protein 1935 C 1248344-1249201 — predicted P-loop-containing kinase 1936 C 1249219-1249713 ptsN nitrogen regulatory IIA protein 1937 C 1249716-1250441 — probable ABC transporter ATP-binding protein 1938 C 1250445-1250963 — conserved hypothetical protein 1939 C 1250944-1251552 — conserved hypothetical protein 1940 C 1251602-1252138 — conserved hypothetical protein 1941 1252227-1253582 pmbA pmbA 1942 1253957-1254496 hpt hypoxanthine phosphoribosyltransferase 1943 1254802-1256124 — predicted Na+/dicarboxylate symporter 1944 C 1256224-1256691 nrdG anaerobic ribonucleoside-triphosphate 1945 reductase activating protein C 1256939-1258669 cydC transport ATP-binding protein CydC 1946 C 1258669-1260429 cydD transport ATP binding protein CydD 1947 C 1260501-1261457 trxB thioredoxin reductase 1948 C 1261531-1262388 — thioredoxin domain-containing protein 1949 C 1262467-1263438 hemH ferrochelatase 1950 C 1263435-1263851 — conserved hypothetical protein 1951 C 1263865-1266948 — conserved FAD/FMN-containing 1952 dehydrogenase 1267323-1268402 ompP5 outer membrane protein P5 1953 1268514-1268876 — conserved glutaredoxin-related protein 1954 C 1268953-1270053 — histidinol-phosphate aminotransferase 2 1955 C 1270141-1271229 serC phosphoserine aminotransferase 1956 1271420-1271764 — conserved hypothetical protein 1957 C 1271804-1272370 — conserved hypothetical protein 1958 C 1272370-1273356 — conserved hypothetical protein 1959 1273398-1273979 trpG2 putative anthranilate synthase component II 1960 1274064-1275218 metK S-adenosylmethionine synthetase 1961 1275513-1276028 sprT SprT 1962 1276079-1276591 opa opacity protein 1963 C 1276627-1276932 — conserved hypothetical protein 1964 C 1277139-1277822 artM arginine transport system permease protein 1965 C 1277822-1278487 artQ arginine transport system permease protein 1966 C 1278491-1279210 artI arginine-binding periplasmic protein 1967 C 1279228-1279959 artP arginine transport ATP-binding protein 1968 C 1280085-1280069 gmhA phosphoheptose isomerase 1969 1280769-1281683 ligA DNA ligase 1970 C 1281712-1282695 dppF dipeptide transport ATP binding protein 1971 C 1282698-1283690 dppD dipeptide transport ATP binding protein 1972 C 1283700-1284587 dppC dipeptide transport system permease protein 1973 C 1284602-1285603 dppB dipeptide transport system permease protein 1974 C 1285693-1287876 uvrD DNA helicase II 1975 C 1288483-1289118 — predicted organic radical activating enzyme 1976 C 1289119-1289544 — predicted 6-pyruval-tetrahydropterin synthase 1977 C 1289537-1290220 — predicted PP-loop superfamily ATPase 1978 1290376-1290519 — conserved hypothetical protein 1979 C 1290570-1291601 ilvE branched chain amino acid amino transferase 1980 1292076-1292969 gcvA glycine cleavage system transcriptional 1981 activator 1292962-1294053 — predicted SAM-dependent methyltransferase 1982 1294089-1295258 sucC succinyl-CoA synthetase beta chain 1983 1295258-1296139 sucD succinyl-CoA synthetase alpha chain 1984 1296260-1296883 — putative translation factor, Sua5 1985 1296931-1289004 rluB ribosomal large subunit pseudouridine synthase B 1986 1298015-1298986 cysB HTH-type transcriptional regulator CysB 1987 C 1299045-1299989 — conserved hypothetical adenine-specific 1988 methylase 1300120-1300623 — conserved hypothetical protein 1989 C 1300701-1302836 pta phosphate acetyltransferase 1990 C 1302904-1304109 ackA acetate kinase 1991 1304326-1304769 — conserved hypothetical protein 1992 1304856-1305347 cvpA colicin C production protein 1993 1305358-1306875 — amidophosphoribosyltransferase 1994 C 1306940-1307830 sulA cell division inhibitor SulA 1995 C 1307830-1308285 argR argininr repressor 1996 1308492-1309427 mdh malate dehydrogenase 1997 C 1309514-1311022 lysS lysyl-tRNA synthetase 1998 C 1311134-1311985 prfB peptide chain release factor 2 1999 1312365-1313054 dsbC thiol:disulfide interchange protein DsbC 2000 1313342-1315069 recJ single stranded DNA specific exonuclease RecJ 2001 1315062-1315769 — conserved hypothetical protein 2002 1315766-1316458 mtnA MTA/SAH nucleosidase 2003 C 1316532-1319279 hup heme utilization protein 2004 C 1319579-1321177 — putative L-Lactate permease 2005 1321367-1321546 — pseudogene for ctidylate kinase 2006 C 1321581-1322585 fbp frustose-1,6-bisphosphatase 2007 1322820-1323629 truA tRNA pseudouridine synthase A 2008 C 1323722-1324738 sapZ Predicted membrane protein 2009 C 1324750-1325559 sapF ABC-type transport system, ATPase component 2010 involved in antimicrobial peptide resistance C 1325559-1326608 sapD ABC-type transport system, ATP binding 2011 component, involved in antimicrobial peptide resistance C 1326612-1327499 sapC ABC-type transport system, permease protein, 2012 involved in antimicrobial peptide resistance C 1327489-1328454 sapB ABC-type transport system, permease protein, 2013 involved in antimicrobial peptide resistance C 1328454-1330148 sapA ABC-type transport system, periplasmic 2014 component, involved in antimicrobial peptide resistance 1330218-1331630 — Predicted ATPase 2015 C 1331667-1334306 Ppc Phosphoenolpyruvate carboxylase 2016 C 1334489-1335499 purR HTH-type transcriptional repressor PurR 2017 1335796-1336689 dapD 2,3,4,5-tetrahydropyridine-2,6-dicarboxylate N- 2018 succinyltransferase C 1336746-1338044 PurA adenylosuccinate synthetase 2019 C 1338173-1338538 — predicted aspartokinase 2020 C 1339386-1339673 rplY 50S ribosomal protein L25 2021 1339841-1340497 — uncharacterized membrane-associate protein 2022 C 1340544-1340960 — conserved hypothetical protein 2023 C 1340991-1341341 — putative translation initiation inhibitor, YjgF 2024 family 1341453-1342169 — conserved hypothetical protein 2025 1342170-1342712 — conserved hypothetical protein 2026 C 1342727-1343185 — HTH-type trancriptional regulator 2027 1343224-1343907 — putative ABC-type Co2+ transport system, 2028 periplasmic component 1343920-1344408 — conserved hypothetical protein 2029 1344408-1345028 cbiM predicted ABC-type cobalt transport system, 2030 permease component 1345028-1345660 — predicted cobalt transport protein 2031 1345662-1346288 cbiO predicted ABC-type cobalt transport system, 2032 ATPase component C 1346395-1347585 aspC aspartate aminotransferase 2033 1347725-1348813 purK phosphoribosylaminoimidazole carboxylase 2034 ATPase subunit C 1348883-1349377 purE phosphoribosylaminoimidazole carboxylase 2035 catalytic subunit 1349548-1349796 hicA ✓ HicA 2036 1349793-1350137 hicB ✓ HicB 2037 1350181-1352790 pepN aminopeptidase N 2038 C 1352862-1353476 ribE riboflavin synthase alpha chain 2039 1353520-1354914 norM probable multidrug resistance protein NorM 2040 1354923-1355639 sfsA sugar fermentation stimulation protein 2041 1355839-1357026 tyrS tyrosyl-tRNA synthase 2042 C 1357090-1358037 prsA ribose phosphate pyrophosphokinase 2043 C 1358039-1358992 ispE 4-diphosphocytidyl-2-C-methyl-D-erythritol 2044 kinase C 1358992-1359621 lolB outer-membrane lipoprotein LolB 2045 C 1359684-1360934 cca tRNA nucleotidyltransferase 2046 C 1360941-1361552 — conserved hypothetical protein 2047 C 1361618-1362880 — putative phosphate permease 2048 C 1362905-1363585 — conserved hypothetical phosphate transport 2049 regulator 1363793-1364248 — predicted membrane protein 2050 1364287-1364574 — conserved hypothetical protein 2051 1364604-1365557 — conserved hypothetical protein 2052 1365547-1366263 — conserved hypothetical protein 2053 1367371-1367538 — ✓ hypothetical protein 2054 1367936-1368052 — ✓ hypothetical protein 2055 1368502 . . . 1368666 — ✓ hypothetical protein 2056 C 1368856-1370808 hmw2C ✓ HMW2C, putative glycosyltransferase involved 2057 in glycosylation of HMW1A and HMW2A C 1371200-1372837 hmw2B ✓ HMW2B, OMP-85-like protein required for 2058 HMW1A and HMW2A secretion C 1372988-1377616 — ✓ HMW2A, high molecular weight adhesin 2 2059 hmw2A 1378041-1379087 — conserved hypothetical protein 2060 1379320-1380696 radA DNA repair protein RadA homolog 2061 1380755-1381255 lrp leucine-responsive regulatory protein 2062 1381257-1384013 ftsK DNa translocase FtsK 2063 1384031-1384648 lolA outer-membrane lipoproteins carrier protein 2064 precursor 1384709-1386049 — predicted ATPase related to the helicase subunit 2065 of the holliday junction resolvase C — ✓ hypothetical protein 2066 1386137 . . . 1387279 C 1387289-1388512 — ✓ modification methylase BepI-like 2067 C 1388767-1390065 aroA 3-phosphoshikimate 1-carboxyvinyltransferase 2068 C 1390065-1390901 purU formyltetrahydrofolate deformylase 2069 C 1390971-1391375 hns DNA-binding protein H-NS homolog 2070 1391779-1393338 — predicted Na+/H+ antiporter 2071 1393406-1393555 — hypothetical protein 2072 1393605-1395326 ilvI acetolactate synthase large subunit 2073 1395326-1395817 ilvH acetolactate synthase small subunit 2074 C 1396111-1397844 argS arginyl-tRNA synthetase 2075 1397924-1398493 — conserved hypothetical protein 2076 1398572-1398793 — hypothetical lipoprotein 2077 1399816-1399280 pcp outer-membrane lipoprotein PCP precursor 2078 C 1399464-1400435 lgtD UDP-0glcNAc-lipooligosaccharide N- 2079 acetylglucosamine glycosyltransferase 1400502 . . . 1400999 — pseudogene for IS1016-V6 protein homolog, 2080 degenerate C 1401073-1402764 pgi glucose-6-phosphate isomerase 2081 C 1402819-1403901 alr alanine racemase 2082 C 1403911-1405425 dnaB replicative DNA helicase 2083 C 1405459-1406895 pykA pyruvate kinase 2084 1407432-1408691 — ✓ prophage CP4-57-like integrase 2085 C 1409305-1409574 — ✓ hypothetical protein 2086 C 1409679-1409849 — ✓ hypothetical protein 2087 C 1409839-1410372 — ✓ hypothetical protein 2088 C 1410444-1410860 — ✓ hypothetical protein 2089 C 1410864-1411541 — ✓ hypothetical protein 2090 C 1411560-1412723 — ✓ modification methylase Bsp6I-like 2091 C 1413329-1414225 rdgC ✓ recombination associated protein 2092 C 1414350-1415696 — ✓ hypothetical protein 2093 C 1415758-1416192 ssb3 ✓ single strand binding protein 2094 C 1416192-1416836 — ✓ hypothetical protein 2095 C 1416817-1417737 — ✓ predicted recombinational DNA repair protein, 2096 RecE pathway C 1417750-1418043 — ✓ hypothetical protein 2097 C 1418040-1418384 — ✓ hypothetical protein 2098 C 1418398-1419135 — ✓ modification methylase DpnIIB-like 2099 C 1419226-1420257 — ✓ hypothetical protein 2100 C 1420247-1420498 — ✓ hypothetical protein 2101 C 1420617-1420928 — ✓ hypothetical protein 2102 C 1421314-1421499 — ✓ hypothetical protein 2103 C 1421660-1422808 — ✓ hypothetical protein 2104 C 1422819-1423679 — ✓ hypothetical protein 2105 C 1423742-1424107 — ✓ hypothetical protein 2106 C 1424109-1424696 — ✓ hypothetical protein 2107 1424852-1425037 — ✓ hypothetical protein 2108 1425068-1425496 — ✓ hypothetical protein 2109 C 1425497-1426006 — ✓ hypothetical protein 2110 C 1426011-1426601 — ✓ hypothetical protein 2111 C 1426598-1427233 — ✓ hypothetical protein 2112 C 1427218-1428000 — ✓ hypothetical protein 2113 C 1428002-1428235 — ✓ hypothetical protein 2114 C 1428281-1428733 — ✓ hypothetical protein 2115 C 1428777-1428983 — ✓ hypothetical protein 2116 1429117-1430085 — ✓ hypothetical protein 2117 1430279-1430605 — ✓ hypothetical protein 2118 1430592-1430996 — ✓ hypothetical protein 2119 1430993-1431382 — ✓ hypothetical protein 2120 1431357-1431587 — ✓ hypothetical protein 2121 1431596-1432555 — ✓ hypothetical protein 2122 1432534-1432953 — ✓ predicted DNA modification methylase 2123 C 1432950-1433171 — ✓ hypothetical protein 2124 1433229-1433597 — ✓ hypothetical protein 2125 1433611-1434927 — ✓ predicted phage terminase large subunit 2126 1434929-1436245 — ✓ hypothetical protein 2127 1436175-1436987 — uncharacterized protein, homolog of phage Mu 2128 protein gp30 1436993-1438066 — ✓ hypothetical protein 2129 1438080-1438499 — ✓ hypothetical protein 2130 1438507-1439508 — ✓ hypothetical protein 2131 1439511-1439699 — ✓ hypothetical protein 2132 1439703-1440053 — ✓ hypothetical protein 2133 1440046-1440504 — ✓ hypothetical protein 2134 1440504-1440863 — ✓ hypothetical protein 2135 1440865-1441374 — ✓ hypothetical protein 2136 1441361-1442434 — ✓ hypothetical protein 2137 1442480-1442905 — ✓ hypothetical protein 2138 1442905-1443387 — ✓ hypothetical protein 2139 1443423-1443575 — ✓ hypothetical protein 2140 1443572-1445989 — ✓ predicted phage-related minor tail protein 2141 C 1446064-1446636 — ✓ hypothetical protein 2142 1446944-1447525 — ✓ hypothetical protein 2143 1447522-1447836 — ✓ hypothetical protein 2144 1447833-1448789 — ✓ hypothetical protein 2145 1448792-1449463 — ✓ hypothetical protein 2146 1449460-1449825 — ✓ hypothetical protein 2147 1449818-1451254 — ✓ hypothetical protein 2148 1451263-1451877 — ✓ hypothetical protein 2149 1451887-1454235 — ✓ probable tail fiber protein 2150 1454247-1454849 — ✓ hypothetical protein 2151 1454846-1455346 — ✓ hypothetical protein 2152 1455412-1455861 — ✓ conserved hypothetical protein 2153 C 1455900-1456163 — ✓ hypothetical protein 2154 1456754-1457884 — pseudogene for putative integrase/recombinase, 2155 degenerate C 1457947-1458228 — hypothetical protein 2156 1458276-1458461 — mu-like prophage protein gp29 2157 1458521-1459189 — conserved hypothetical protein 2158 1459291-1459437 — hypothetical protein 2159 1459470-1460006 — conserved hypothetical protein 2160 1460207-1460653 — ✓ hypothetical protein 2161 1460653-1460829 — ✓ hypothetical protein 2162 1430841-1461500 — ✓ hypothetical protein 2163 1461500-1461676 — ✓ hypothetical protein 2164 1461689-1462246 — ✓ hypothetical protein 2165 1462243-1462419 — ✓ hypothetical protein 2166 1462432-1462983 — ✓ hypothetical protein 2167 1463259-1464344 prfA peptide chain release factor 1 2168 1464367-1464837 — conserved hypothetical protein 2169 1464837-1465715 hemK HemK 2170 1465715-1466518 — conserved hypothetical protein 2171 1466533-1467387 kdsA 2-dehyrdro-3-deoxyphosphooctonate aldolase 2172 1467447-1468394 — putative 2-hydroxyacid dehydrogenase 2173 1468394-1469575 lolC lipoprotein releasing system transmembrane 2174 protein 1469587-1470879 bioA adenosylmethionine-8-amino-7-oxononanoate 2175 aminotransferase 1470888-1472027 bioF 8-amino-7-oxononanoate synthase 2176 1472037-1472684 — conserved hypothetical protein 2177 1472672-1473454 bioC putative biotin synthesis protein BioC 2178 1473464-1474105 bioD-B probable dethiobiotin synthetase 2 2179 1474187-1474870 lolD lipoprotein releasing system ATP-binding 2180 protein LolD 1474870-1476120 lolE ABC-type transport system, involved in 2181 lipoprotein release, permease component 1476339-1477427 aroG phospho-2-dehyrdro-3-deoxyheptonate aldolase 2182 C 1477497-1477937 impA impA 2183 1478222-1479466 — predicted Na+/serine symporter 2184 1479588-1480196 — putative NAD(P)H oxidoreductase 2185 1480196-1480750 — predicted component of anaerobic 2186 dehydrogenases C 1480797-1481351 — predicted nitroreductase 2187 1481449-1483296 sppA protease IV 2188 C 1483334-1484131 licD phosphorylcholine transferase 2189 C 1484131-1484832 licC LicC 2190 C 1484829-1485707 licB LicB 2191 C 1485707-1486666 licA LicA 2192 C 1486754-1488766 — predicted glycine/D-amino acid oxidases, 2193 deaminating 1488913-1490133 fabB 3-oxacyl-[acyl-carrier-protein] synthase I 2194 C 1490196-1490459 rrxA glutaredoxin 2195 1490576-1491484 rimK probable ribosomal protein S6 modification 2196 protein C 1495122-1492736 gltS sodium/glutamate symport carrier protein 2197 C 1492778-1495021 parC topoisomerase IV subunit A 2198 C 1495088-1496986 parE topoisomerase IV subunit B 2199 C 1497060-1497995 htrB lipid A biosynthesis lauroyl acyltransferase 2200 1498092-1499522 rfaE ADP-heptose synthase 2201 C 1499886-1499981 — pseudogene for Type I restriction enzyme R 2202 protein C 1500193-1500549 — ✓ hypothetical protein 2203 1500660-1501064 uupA2 ABC transporter ATP-binding protein 2204 1501061-1501621 uppB ABC transporter ATP-binding protein 2205 C 1501649-1502338 — putative carbonic anhydrase 2206 C 1502414-1503922 asnS asparaginyl-tRNA synthetase 2207 1504044-1504517 ribH 6,7-dimethyl-8-ribityllumazine synthase 2208 1504521-1504955 nusB N utilization substance protein B 2209 1505022-1506008 thiL thiamine-monophosphate kinase 2210 1506005-1506496 pgpA phosphatidylglycerophosphatase A 2211 1506496-1507125 — predicted threonine efflux protein 2212 1507142-1507954 dapB dihydrodipicolinate reductase 2213 C 1507949-1508197 — conserved hypothetical ferredoxin-like protein 2214 1508243-1508983 — conserved hypothetical protein 2215 1509132-1510121 pheS ohenylalanyl-tRNA synthetase alpha chain 2216 1510155-1512542 pheT ohenylalanyl-tRNA synthetase beta chain 2217 1512544-1512834 himA integration host factor alpha-subunit 2218 1512887-1513372 — conserved hypothetical lipoprotein 2219 1513381-1514391 — ✓ hypothetical protein 2220 1514450-1514905 — ✓ putative 5′(3′)-deoxyribonucleotidase 2221 1514889-1515599 — ✓ NAD-dependent deacetylase 2222 C 1515648-1516670 — ✓ hypothetical protein 2223 1516865-1517065 ftsK2 ✓ DNA translocase ftsK 2224 1517062-1517907 — ✓ NAD-dependent deacetylase sirtuin 5 2225 1517907-1518668 — ✓ hypothetical protein 2226 1518746-1519720 — ✓ hypothetical protein 2227 1519797-1519964 — ✓ hypothetical protein 2228 1520134-1521849 — ✓ predicted serine/threonine protein phosphatase 2229 family protein 1521869-1522306 — predicted arylsulfatase A-like enzyme 2230 1522388-1523200 — predicted enzyme related to aldose 1-epimerase 2231 1523468-1523875 infC translation initiation factor IF-3 2232 1524014-1524283 rpmI 50S ribosomal protein L35 2233 1524349-1524702 rplT 50S ribosomal protein L20 2234 1524815-1528450 recB exodeoxyribonuclease V beta chain 2235 1528450-1530372 recD exodeoxyribonuclease V alpha chain 2236 C 1530441-1530887 — conserved hypothetical protein 2237 1530984-1532789 lonB putative protease Lahomolog, predicted ATP- 2238 dependent protease 1532958-1533491 fabA 3-hydroxydecanoyl-[acyl-carrier-protein] 2239 dehydratase 1533727-1534455 — conserved hypothetical protein 2240 1534452-1534589 — conserved hypothetical protein 2241 1534725-1534994 rpsO 30S ribosomal protein S15 2242 C 1535066-1535437 — conserved hypothetical transposase-like protein 2243 C 1535434-1535550 — conserved hypothetical protein 2244 C 1535766-1537205 dacB penicillin-binding protein 4 precursor 2245 1537369-1537845 greA transcription elongation factor GreA 2246 C 1537884-1538183 — predicted RNA-binding protein containing KH 2247 domain, possible ribosomal protein 1538310-1538939 ftsJ ribosomal RNA large subunit methyltransferase J 2248 1539030-1540937 FTSh cell division protein FtsH homolog 1 2249 1541049-1541879 folP dihydropteroate synthase 2250 1541912-1543249 mrsA predicted phosphomannomutase 2251 1543308-1543802 sixA phosphohistidine phosphatase SixA homolog 2252 1544004-1544579 — ✓ conserved hypothetical protein 2253 C 1544657-1546021 — conserved hypothetical protein 2254 1546248-1546388 — hypothetical protein 2255 1546382-1546534 — hypothetical protein 2256 1546609-1546767 — pseudogene for surface protein autotransporter 2257 domain 1546843-1547439 — possible RNA polymerase sigma factor 24 2258 1547449-1547622 — hypothetical protein 2259 C 1547717-1548619 — ✓ hypothetical protein 2260 1548755-1549816 msaB peptide methionine sulfoxide reductase 2261 MsrA/MsrB 1549828-1550469 — conserved hypothetical cytochrome c-type 2262 biogenesis protein 1550514-1550984 — conserved hypothetical protein 2263 C 1551166-1551489 — conserved hypothetical protein 2264 C 1551556-1552287 moeB molybdopterin biosynthesis protein MoeB 2265 C 1552291-1553505 moeA molybdopterin biosynthesis protein MoeA 2266 1553643-1554299 folE GTP cyclohydrolase I 2267 1554303-1554626 — conserved hypothetical protein 2268 1554729-1555457 bioD-A probable dethiobiotin synthetase 1 2269 C 1555546-1556424 metF 5,10-methylenetetrahydrofolate reductase 2270 1556887-1557315 rplM 50S ribosomal protein L13 2271 1557332-1557724 rpsI 30S ribosomal protein S9 2272 1557905-1558543 sspA stringent starvation protein A 2273 1558543-1558995 sspB stringent starvation protein B 2274 C 1559031-1560908 dxs 1-deoxy-D-xylulose 5-phosphate synthase 2275 C 1560950-1561840 ispA geranyltranstransferase 2276 C 1561840-1562094 xseB exodeoxyribonuclease VII small subunit 2277 1562253-1563710 thiI predicted thiamine biosynthesis ATP 2278 pyrophosphatase 1563725-1564045 — conserved hypothetical protein 2279 1564039-1454758 truC tRNA pseudouridine synthase C 2280 1564751-1564909 — conserved hypothetical protein 2281 1565058-1565276 cspD cold shock-like protein CspD 2282 C 1565415-1565891 — conserved hypothetical protein 2283 1565950-1566903 usg predicted aspartate-semialdehyde 2284 dehydrogenase C 1567047-1567853 trpA tryptophan synthase alpha chain 2285 C 1567853-1569046 trpB tryptophan synthase beta chain 2286 C 1569063-1569821 — conserved hypothetical oxidoreductase 2287 1570001-1571035 purM phosphoribosylformylglycinamidine cyclo- 2288 ligase 1571090-1571728 purN phosphoribosylglycinamide formyltransferase 2289 C 1571812-1572648 — predicted ABC-type transport system protein, 2290 periplasmic component C 1572846-1573868 uspE universal stress protein E 2291 C fnr fumarate and nitrate reduction regulatory 2292 1573897 . . . 1574670 protein 1575288-1575644 — putative integrase/recombinase 2293 C 1575529-1575918 — conserved hypothetical protein 2294 C 1576223-1576474 — predicted phage anti-repressor protein 2295 C 1576812-1578083 — ✓ hypothetical protein 2296 1578684-1578887 — ✓ hypothetical protein 2297 C 1578865-1579023 — ✓ hypothetical protein 2298 C 1579351-1579572 — ✓ hypothetical protein 2299 C 1580499-1580834 — ✓ hypothetical protein 2300 C 1580837-1581916 — ✓ hypothetical protein 2301 C 1581968-1582666 — ✓ predicted transcriptional regulator 2302 1582777-1582965 — ✓ hypothetical protein 2303 1583014-1583454 — ✓ hypothetical protein 2304 1583503-1584177 — ✓ hypothetical protein 2305 1584174-1584932 — ✓ hypothetical protein 2306 1584917-1585558 — ✓ hypothetical protein 2307 1585555-1585779 — ✓ hypothetical protein 2308 1585816-1586232 ninB ✓ putative recombination protein NinB 2309 1586500-1586889 ninG putative recombination protein NinG homolog 2310 1586909-1587538 — ✓ hypothetical protein 2311 1587662-1587940 — conserved hypothetical protein 2312 C 1587990-1588967 — ✓ hypothetical protein 2313 1589196-1589981 — conserved hypothetical protein 2314 1590012-1590194 — hypothetical protein 2315 1590318-1590674 — conserved hypothetical protein 2316 1590754-1591245 — conserved hypothetical protein 2317 1591238-1591561 — hypothetical protein 2318 1591473-1591754 — conserved hypothetical protein 2319 C 1591756-1592034 — ✓ hypothetical protein 2320 1592079-1592591 — conserved hypothetical protein 2321 1592578-1593921 — predicted phage terminase large subunit 2322 1593923-1595257 — conserved hypothetical protein 2323 1595286-1596467 — ✓ uncharacterized protein, homolog of phage Mu 2324 protein gp30 1596613-1596789 — conserved hypothetical protein 2325 1596953-1597972 pyrD dihyrdoorotate dehydrogenase 2326 1597972-1598796 trpH TrpH 2327 C 1598895-1599503 — conserved hypothetical protein 2328 C 1599631-1601025 fumC fumurate hydratase class II 2329 C 1601175-1602113 — ✓ putative glycosyl transferase, glycosyl 2330 transferase family 8 protein 1602322-1602666 — pseudogene for usg protein homolog 2331 1602769-1603203 holC DNA polymerase III, chi subunit 2332 1603288-1603458 — conserved hypothetical protein 2333 1603471-1603845 — conserved hypothetical protein 2334 1603873-1604040 — hypothetical protein 2335 1604067-1604381 — conserved hypothetical protein 2336 1604425-1604826 — ✓ hypothetical protein 2337 1604878-1607742 valS valyl-tRNA synthetase 2338 C 1607777-1607884 — hypothetical protein 2339 C 1608172-1609605 trpC tryptophan biosynthesis protein trpCF 2340 C 1609643-1610644 trpD anthranilate phosphoribosyltransferase 2341 C 1610697-1611083 — conserved hypothetical protein 2342 C 1611132-1611719 trpG anthranilate synthase component II 2343 C 1611732-1613288 trpE anthranilate synthase component I 2344 C 1613390-1614300 — ✓ pseudogene for conserved hypothetical 2345 glycosyltransferase C 1614423-1614920 ftnB ferritin-like protein 2 2346 C 1614936-1615484 ftnA ferritin-like protein 1 2347 1615927-1616946 pstS phosphate-binding periplasmic protein 2348 precursor PstS 1617038-1617985 pstC phosphate transport system permease protein 2349 PstC 1617987-1618835 pstA phoaphate transport system permease proteain 2350 PstA 1618845-1619612 pstB phosphate import ATP-binding protein PstB 2351 1619709-1620404 phoB phosphate regulon transcriptional regulatory 2352 protein PhoB 1620401-1621678 phoR phosphate regulon sensor protein PhoR 2353 C 1622370-1623791 sbcB exodeoxyribonuclease I 2354 C 1623804-1624679 — conserved hypothetical protein 2355 C 1624683-1625591 — conserved hypothetical protein 2356 C 1625661-1630193 — cell division protein MukB 2357 C 1630193-1630927 — cell division protein MukE 2358 C 1630931-1631989 haeIIR ✓ type II restriction exzyme HaeII 2359 C 1631992-1632948 haeIIM ✓ modification methylase HaeII 2360 C 1632961-1634388 mukF MukF homolog 2361 1634445-1635377 — predicted ATPase of the PP-loop superfamily 2362 implicated in cell cycle control 1635379-1635708 — predicted dissimilatory sulfite reductase, 2363 desulfoviridin, gamma subunit 1635816-1636025 mop probable molybdenum-pterin binding protein 2364 1636209-1638621 — pseudogene for conserved hypothetical protein, 2365 denegerate 1638725-1641505 pqqL probable zinc protease 2366 C 1641628-1643559 thrS threonyl-tRNA synthetase 2367 1643843-1644427 acpD probable acyl carrier protein phosphodiesterase 2368 C 1644504-1647110 topA DNA topoisomerase I 2369 1647206-1648123 — putative HTH-type transcriptional regulator 2370 C 1648187-1649611 pntB NAD(P) transhydrogenase subunit beta 2371 C pntA NAD(P) transhydrogenase subunit alpha 2372 1649622 . . . 1651160 C 1651421-1653886 glgP glycogen phosphorylase 2373 C 1654133-1655563 glgA glycogen synthase 2374 C 1655671-1657005 glgC glucose-1-phosphate adenylyltransferase 2375 C 1656955-1658971 glgX glycogen operon protein GlgX 2376 C 1659070-1661262 glgB 1,4-alpha-glucan branching enzyme 2377 C 1661272-1663371 malQ 4-alpha-glucanotransferase 2378 C 1663433-1663897 — conserved hypothetical protein 2379 C 1663963-1665636 glnS glutaminyl-tRNA synthetase 2380 C 1666056-1667531 cafA ribonuclease G 2381 1667647-1669161 putP sodium/proline symporter 2382 1669158-1670123 — conserved hypothetical protein 2383 C 1670166-1671044 cdd cytiding deaminase 2384 1671318-1671800 — conserved hypothetical DNA-binding ferritin- 2385 like protein C 1672372-1673610 pepT peptidase T 2386 1673880-1675025 potA spermidine/putrescine transport ATP-binding 2387 protein 1675009-1675869 potB spermidine/putrescine transport permease 2388 protein 1675869-1676639 potC spermidine/putrescine transport system 2389 permease protein 1676686-1677852 potD1 spermidine/putrescine-binding periplasmic 2390 protein 1 precursor C 1677933-1679876 uupA ABC transporter ATP-binding protein 2391 C 1679970-1681355 — deoxyguanosinetriphosphate 2392 triphosphohydrolase-like protein C 1681448-1682143 — putative effector of murein hydrolase 2393 C 1682162-1682584 — putative effector of murein hydrolase 2394 1682699-1683181 — predicted micrococcal nuclease-like protein 2395 1683183-1684382 nifS predicted selenocysteine lyase 2396 1684379-1684759 — predicted SufE protein probably involved in Fe—S 2397 center assembly 1684750-1685547 — Zn-ribbon-containing, possible nucleic-acid 2398 binding protein 1685591-1686430 — predicted enzyme related to GTP 2399 cyclohydrolase I C 1686463-1687587 tyrA T-protein 2400 C 1687677-1688597 truB tRNA pseudouridine synthase B 2401 C 1688597-1688983 rbfA ribosome-binding factor A 2402 1689148-1690818 hsdM3 putative type I restriction enzyme HindVIIP M 2403 protein 1690811-1692076 hsdS3 ✓ putative type I restriction enzyme HindVIIP 2404 specificity protein 1692069-1693115 — ✓ hypothetical protein 2405 1693117-1696284 hsdR3 putative type I restriction enzyme HindVIIP R 2406 protein C 1696363-1698897 infB translation initiation factor IF-2 2407 C 1698909-1700396 nusA transcriptional elongation protein NusA 2408 C 1700413-1700868 — conserved hypothetical protein 2409 C 1702529-1703293 — ✓ hypothetical protein 2410 C 1703297-1703824 — ✓ hypothetical protein 2411 C 1703834-1704973 — ✓ hypothetical protein 2412 C 1704990-1705358 — ✓ hypothetical protein 2413 C 1705372-1706193 — ✓ hypothetical protein 2414 C 1706250-1706753 — ✓ hypothetical protein 2415 C 1706750-1707352 — ✓ hypothetical protein 2416 C 1707364-1709886 — ✓ probable tail fiber protein 2417 C 1709895-1710431 — ✓ predicted bacteriophage P2-related tail 2418 formation protein gpI C 1710421-1711335 — ✓ predicted phage-related baseplate assembly 2419 protein C 1711332-1711670 — ✓ predicted baseplate assembly protein W 2420 C 1711672-1712271 — ✓ predicted phage P2-like baseplate assembly 2421 protein C 1712375-1712803 — ✓ hypothetical protein 2422 C 1712812-1713369 — ✓ hypothetical protein 2423 C 1713467-1713919 — ✓ hypothetical protein 2424 C 1713897-1714097 — ✓ hypothetical protein 2425 C 1714141-1716105 — ✓ predicted phage-related tail protein 2426 C 1716148-1716915 — ✓ hypothetical protein 2427 1716960-1717298 — ✓ hypothetical protein 2428 C 1717309-1717491 — ✓ hypothetical protein 2429 1717595-1717942 — ✓ hypothetical protein 2430 C 1717943-1718404 — ✓ probable bacteriophage tail completion protein 2431 gpS homolog C 1718404-1718790 — ✓ probable bacteriophage tail completion protein 2432 gpR homolog C 1718840-1718947 — ✓ hypothetical protein 2433 C 1718980-1719102 — ✓ hypothetical protein 2434 C 1719099-1719314 — ✓ DnaK suppressor protein, bacteriophage PSP3 2435 gp34 homolog C 1719489-1719839 — ✓ hypothetical protein 2436 C 1719824-1720342 — ✓ predicted phage-related lysozyme 2437 1720335 . . . 1720556 — ✓ hypothetical protein 2438 C 1720558-1720767 — ✓ hypothetical protein 2439 C 1720767-1721273 — ✓ hypothetical protein 2440 C 1721567-1722217 — ✓ predicted terminase, endonuclease subunit 2441 C 1722229-1723278 — ✓ predicted major capsid protein 2442 C 1723299-1724114 — ✓ predicted capsid scaffolding protein 2443 1724279-1726060 — ✓ terminase, ATPase subunit 2444 1726070-1727080 — ✓ predicted portal vertex protein 2445 C 1727788-1728462 siaB acylneuraminate cytidylyltransferase 2446 1728576-1729238 — putative NAD(P)H nitroreductase 2447 C 1729285-1730445 mrp Mrp 2448 1730548-1732596 metG methionyl-tRNA synthetase 2449 1732707-1733567 tehB tellurite resistance protein 2450 C 1733611-1734312 gloB probable hydroxyacylglutathione hydrolase 2451 C 1734364-1735170 — conserved hypothetical protein 2452 1735406-1735882 — conserved hypothetical protein 2453 1736019-1737782 — conserved hypothetical protein 2454 1738382-1741024 gyrA DNA gyrase subunt A 2455 C 1741079-1742155 metX homoserine O-acetyltransferase 2456 1742259-1742993 sanA SanA 2457 C 1743281-1744594 folC folypolyglutamate synthase 2458 C 1744587-1745477 accD acethy-coenzyme A carboxylase carboxyl 2459 transferase subunit beta 1745671-1747062 htoA probable periplasmic serine protease do/HhoA- 2460 like precursor C 1747115-1750555 mfd transcription-repair coupling factor 2461 C 1750657-1750830 — conserved hypothetical protein 2462 C 1750827-1752797 — predicted P-loop ATPase fused to an 2463 acetyltransferase C 1752802-1753143 — conserved hypothetical protein 2464 C 1753205-1754875 — ABC transported ATP-binding protein 2465 C 1755038-1755361 — predicted plasmid maintenance system antidote 2466 protein C 1755372-1755677 — predicted plasmid maintenance system killer 2467 protein 1756002-1756622 — predicted ABC-type transport system, 2468 periplasmic component 1756625-1757593 — predicted ABC-type transport system, permease 2469 component C 1758207-1760246 uvrB UvrABC system protein B 2470 C 1760276-1762201 — predicted phosphoglycerol transferase-like 2471 protein C 1762442-1764712 mao2 NADP-dependent malic enzyme 2472 1764910-1765530 — possible polysaccharide biosynthesis protein 2473 1765621-1766319 rsuA ribosomal small subunit pseodouridine synthase A 2474 1766321-1767517 bcr bicyclomycin resistance protein 2475 C 1767549-1768349 — conserved hypothetical protein 2476 1768447-1769109 — predicted membrane protein 2477 1769130-1770383 proA gamma-glutamyl phosphate reductase 2478 C 1770462-1771607 dnaJ chaperone protein DnaJ 2479 C 1771694-1773601 dnaK chaperone protein DnaK 2480 1773868-1775310 — conserved hypothetical protein 2481 C 1775467-1775925 mgsA methylglyoxal synthase 2482 1776225-1778885 aceE pyruvate dehydrogenase E1 component 2483 1778948-1780645 aceF dihydrolipoamide acetyltransferase component 2484 of pyruvate dehydrogenase complex 1780759-1782183 lpdA dihydrolipoamide dehudrogenase 2485 C 1782230-1782337 — hypothetical protein 2486 1782343-1782885 apt adenine phosphoribosyltransferase 2487 1782897-1784963 dnzX DNA polymerase III subunit gamma/tau 2488 1785103-1785729 upp uracil phosphoribosyltransferase 2489 1785833-1787077 uraA probable uracil permease 2490 1787143-1787838 — predicted ATPase involved in DNA replication 2491 initiation C 1787905-1788186 — predicted translation initiation factor 1-like 2492 proterin C 1788193-1788885 pyrF orotidine 5′-phosphate decarboxylase 2493 C 1788909-1790099 — predicted N-acetylglucosaminyl transferase 2494 C 1790099-1790392 — predicted membrane protein 2495 C 1790467-1790751 ihfB integration host factor beta subunit 2496 C 1790874-1792523 rpsA 30S ribosomal protein S1 2497 C 1792626-1793420 cmk ctidylate kinase 2498 1793766-1794641 — conserved hypothetical pyridoxine biosynthesis 2499 enzyme 1794642-1795220 — predicted glutamine amidotransferase involved 2500 in pyridoxine biosynthesis C 1795769-1797463 dld D-lactate dehydrogenase 2501 1797723-1797833 — conserved hypothetical protein 2502 C 1798368-1798919 nlpC probable lipoprotein NlpC 2503 C 1799032-1800480 tldD TldD 2504 C 1800582-1801433 — predicted methyltransferase 2505 1801505-1803232 — putative lipoprotein 2506 1803232-1803591 — predicted endonuclease distantly related to 2507 archael holliday junction resolvase 1803604-1804188 — predicted phosphoheptose isomerase 2508 1804245-1804826 — predicted periplasmic or secreted lipoprotein 2509 1805081-1807351 nrdA ribonucleoside-diphosphate reductase alpha 2510 chain 1807493-1808623 nrdB ribonucleoside-diphosphate reductase beta 2511 chain C 1808739-1809968 sucB dihydrolipoamide succinyltransferase 2512 component of 2-oxoglutarate dehydrogenase complex C 1810071-1812923 sucA 2-oxoglutarate dehydrogenase E1 component 2513 C 1813081-1813719 — predicted Zn-dependent hydrolase-like protein, 2514 including glyoxylases C 1813729-1814484 — conserved putative deoxyribonuclease 2515 C 1814536-1815096 — conserved hypothetical protein 2516 C 1815112-1816581 — conserved hypothetical protein 2517 C 1816655-1818742 prc tail-specific protease precursor 2518 C 1818764-1819402 proQ predicted activator of osmoprotectant 2519 transporter PropP 1819587-1820837 — paraquat-inducible protein A-like protein 2520 1820821-1823466 — paraquat-inducible protein B-like protein 2521 C 1823521-1823973 moaE molybdopterin converting factor subunit 2 2522 C 1823974-1824219 moaD molybdopterin converting factor subunit 1 2523 C 1824221-1824703 moaC molybdenum cofactor biosynthesis protein C 2524 C 1824795-1825808 moaA molybdenum cofactor biosynthesis protein A 2525 1826253-1826726 — predicted regulator of cell morphogenesis and 2526 NO signaling 1826726-1826923 predicted regulator of cell morphogenesis and 2527 NO signaling 1827460-1728473 — probable phosphosugar isomerase Hi1678 2528 1828483-1829025 yrbI 3-deoxy-D-manno-octulosonate 8-phosphate 2529 phosphatase 1829456-1833934 hmw1A ✓ HMW1A, high molecular weight adhesin 1 2530 1834085-1835722 hmw1B ✓ HMW1B, OMP-85-like protein required for 2531 secretion of HMW1A and HMW2A 1835916-1837868 hmw1C ✓ HMW1C, putative glycosyltransferase involved 2532 in glycosylation of HMW1A and HWM2A C 1837945-1840101 — predicted membrane protein 2533 C 1840169-1840834 — conserved hypothetical protein 2534 1841032-1842093 sohB possible protease SohB 2535 1842377-1842955 rnfA predicted NADH:ubiquinone oxidoreductase, 2536 subunit RnfA 1843043-1843624 rnfB predicted NADH:ubiquinone oxidoreductase, 2537 subunit RnfB 1843625-1845622 rnfC predicted NADH:ubiquinone oxidoreductase, 2538 subunit RnfC 1845752-1846828 rnfD predicted NADH:ubiquinone oxidoreductase, 2539 subunit RnfD 1846828-1847451 rnfG predicted NADH:ubiquinone oxidoreductase, 2540 subunit RnfG 1847453-1848160 — predicted NADH:ubiquinone oxidoreductase, 2541 subunit RnfE 1848282-1848917 nth endonuclease III 2542 1848976-1850349 — predicted Na+-dependent transporters of the 2543 SNF family C 1850373-1851428 modC molybdenum import ATP-binding protein 2544 C 1851415-1852104 modB molybdenum transport system permease protein 2545 C 1852278-1853042 modA molybdate-binding periplasmic protein 2546 1853180-1853947 mode Transcriptional regulator ModE 2547 1854057-1854860 lsgF Putative UDP-galactose-lipooligosaccharide 2548 galactosyltransferase C 1854862-1855746 lsgE Putative UDP-galactose-lipooligosaccharide 2549 galactosyltransferase C 1855758-1856531 lsgD Putative UDP-glcNAc-lipooligosaccharide 2550 N-acetylglucosaminyl glycosyltransferase C 1856543-1857604 lsgC Putative UDP-galactose--lipooligosaccharide 2551 galactosyltransferase C 1857606-1858520 lsgB CMP-N-acetylneuraminate-beta-galactosamide- 2552 alpha-2,3-sialytransferase C 1858517-1859722 lsgA putative lipooligosaccharide flippase 2553 1859857-1860600 — conserved hypothetical protein 2554 C 1860823-1863092 metE pseudogene for 5- 2555 nethyltetrahydropteroytrigultamate— homocysteine methyltransferase C 1863286-1864362 — predicted permease 2556 C 1864359-1865462 — predicted permease 2557 1865570-1867045 pepA cytosol aminopeptidase 2558 C 1867081-1869090 — predicted choline-glycine betaine transporter 2559 C 1869385-1870740 qseC sensor protein QseC 2560 C 1870737-1871402 qseB transcriptional regulatory protein QseB 2561 C 1871466-1871831 — conserved hypothetical protein 2562 C 1871946-1872966 pmi ✓ pseudogene for mannose-6-phosphate 2563 isomerase C 1873336-1873836 crr PTS system, glucose-specific IIA component 2564 C 1873896-1875623 ptsI Phosphoenolpyruvate-protein 2565 phosphotransferase C 1875703-1875960 ptsH Phosphocarrier protein HPr 2566 C 1876119-1877159 — probable GTPase 2567 1877230-1877778 orn oligoribonuclease 2568 C 1878178-1879245 wecA undecaprenyl phosphate 2569 C 1879320-1881911 glnD [protein-PII] uridylyltransferase 2570 C 1882005-1882811 map methionine aminopeptidase 2571 1882947-1883291 — conserved hypothetical protein 2572 1883293-1883646 — conserved hypothetical protein 2573 1883653-1885998 mrcB penicillin-binding protein 1B 2574 1886123-1887043 purC phosphoribosylaminoimidazole- 2575 succinocarboxamine synthase 1887199-1888533 argG argininosuccinate synthase 2576 C 1888604-1889797 — Mn2+ and Fe2+ transporter of the NRAMP 2577 family C 1889841-1890578 — conserved hypothetical protein 2578 C 1890565-1891494 — predicted allophanate hydrolase subunit 2 2579 C 1891491-1892132 — predicted allophanate hydrolase subunit 1 2580 C 1892394-1894373 rnb exoribonuclease II 2581 C 1894471-1895358 fabI enoyl-[acyl-carrier-protein] reductase [NADH] 2582 1895438-1897021 prfC peptide chain release factor 3 2583 C 1897097-1897330 — conserved hypothetical protein 2584 C 1897433-1897762 — predicted branched chain amino acid permease 2585 C 1897759-1898493 — predicted branched chain amino acid permease 2586 C 1898503-1899432 metR HTH-type transcriptional regulator MetR 2587 C 1905763-1906908 lldD L-lactate dehydrogenase 2588 C 1907120-1907929 murI glutamate racemase 2589 C 1907959-1910040 recG ATP-dependent DNA helicase 2590 C 1910037-1912151 spoT guanosine-3′,5′-bis 3′pyrophosphohydrolase 2591 C 1912224-1912490 rpoZ DNA directed RNA polymerase omega chain 2592 C 1912553-1913179 gmk guanylate kinase 2593

TABLE 7 Gene Products of the NTHi Genome SEQ Gene ID Name Product Name NO: gapA glyceraldehyde 3-phosphate dehydrogenase 2594 — putative long-chain-fatty-acid--CoA ligase 2595 — conserved hypothetical protein 2596 — conserved hypothetical protein 2597 fdhD FdhD protein homolog 2598 fdxG formate dehydrogenase major subunit 2599 fdxH formate dehydrogenase, iron-sulfur subunit 2600 fdxI formate dehydrogenase, cytochrome B556 subunit 2601 fdhE FdhE homolog 2602 rimI ribosomal-protein-alanine acetyltransferase 2603 holD DNA polymerase III, psi subunit 2604 rsmC ribosomal RNA small subunit methyltransferase C 2605 era GTP-binding protein era homolog 2606 rnc ribonuclease III 2607 lepB Signal peptidase I 2608 lepA GTP-binding protein LepA 2609 — conserved hypothetical acid-induced glycyl 2610 radical enzyme ung uracil-DNA glycosylase 2611 — conserved hypothetical protein 2612 — conserved hypothetical protein 2613 citG CitXG 2614 citF citrate lyase alpha chain 2615 citE citrate lyase beta chain 2616 citD citrate lyase acyl carrier protein 2617 citC [citrate [pro-3S]-lyase] ligase 2618 lipA Lipoic acid synthetase 2619 lipB lipoate-protein ligase B 2620 — conserved hypothetical protein 2621 dacA penicillin-binding protein 5 precursor 2622 rlpA RlpA-like protein 2623 mrdB Rod shape-determining protein RodA 2624 mrdA penicillin-binding protein 2 2625 — conserved hypothetical protein 2626 — conserved hypothetical protein 2627 — conserved hypothetical membrane protein 2628 — conserved hypothetical ABC transporter ATP-binding 2629 protein mreB rod shape-determining protein MreB 2630 mreC rod shape-determining protein MreC 2631 mreD rod shape-determining protein MreD 2632 — conserved hypothetical protein 2633 xthA exodeoxyribonuclease III 2634 rluA2 conserved hypothetical pseudouridine synthase 2635 — conserved hypothetical membrane protein 2636 — conserved hypothetical FtsH-interacting integral 2637 membrane protein — conserved hypothetical protein 2638 phnA PhnA homolog 2639 eda KHG/KDPG aldolase 2640 uxuC uronate isomerase 2641 — putative oxidoreductase 2642 — putative TRAP-type C4-dicarboxylate transport 2643 system, large permease component — putative TRAP-type C4-dicarboxylate transport 2644 system, small permease component — putative TRAP-type C4-dicarboxylate transport 2645 system, periplasmic component — conserved hypothetical zinc-type alcohol 2646 dehydrogenase-like protein uxuR Uxu operon regulator 2647 uxuA mannonate dehydratase 2648 — putative membrane protein TerC 2649 uvrC UvrABC system protein C 2650 kdsB 3-deoxy-D-manno-octulosonic acid 2651 cytidylyltransferase lpxK tetraacyldisaccharide 4′-kinase 2652 msbA lipid A export ATP-binding protein MsbA 2653 rec2 recombination protein 2 2654 dksA DnaK suppressor protein 2655 pcnB probable poly polymerase 2656 folK 2-amino-4-hydroxy-6-hydroxymethyl- 2657 dihydropteridine pyrophosphokinase — conserved hypothetical protein 2658 amiB probable N-acetylmuramoyl-L-alanine amidase AmiB 2659 precursor mutL DNA mismatch repair protein MutL 2660 miaA tRNA delta-isopentenylpyrophosphate transferase 2661 glnE glutamate-ammonia-ligase adenylyltransferase 2662 recN DNA repair protein RecN 2663 ppnK probable inorganic polyphosphate/ATP-NAD kinase 2664 grpE grpE 2665 — conserved hypothetical protein 2666 — conserved hypothetical protein 2667 nrdD anaerobic ribonucleoside-triphosphate reductase 2668 tesB Acyl-CoA thioesterase II 2669 cysS cysteinyl-tRNA synthetase 2670 ppiB peptidyl-prolyl cis-trans isomerase B 2671 — conserved hypothetical protein 2672 — putative deoxyribonuclease 2673 — hypothetical protein 2674 — hypothetical protein 2675 trxA thioredoxin 2676 ddh 2-hydroxyacid dehydrogenase homolog 2677 metB cystathionine gamma-synthase 2678 — predicted ATPase involved in chromosome 2679 partitioning dnaB2 replicative DNA helicase 2680 — conserved hypothetical protein 2681 — conserved hypothetical protein 2682 — conserved hypothetical protein 2683 — conserved hypothetical protein 2684 — conserved hypothetical protein 2685 ssb2 Single-strand binding protein 2686 — conserved putative lipoprotein 2687 — 1conserved hypothetical protein 2688 topB2 DNA topoisomerase III 2689 — conserved hypothetical protein 2690 — hypothetical protein 2691 — conserved hypothetical protein 2692 — hypothetical protein 2693 radC2 putative DNA repair radC-like protein 2694 — conserved hypothetical protein 2695 — conserved hypothetical protein 2696 pilL conserved putative lipoprotein 2697 — conserved putative exported protein 2698 — conserved putative exported protein 2699 — conserved putative exported protein 2700 — conserved hypothetical protein 2701 — conserved putative membrane protein 2702 — conserved putative membrane protein 2703 — conserved putative membrane protein 2704 — conserved putative membrane protein 2705 — conserved putative exported protein 2706 — conserved hypothetical protein 2707 — conserved putative exported protein 2708 — conserved putative lipoprotein 2709 — conserved hypothetical protein 2710 — conserved hypothetical protein 2711 — conserved hypothetical membrane protein 2712 — conserved putative membrane protein 2713 — conserved hypothetical membrane protein 2714 tnpA transposon Tn3 transposase 2715 — hypothetical protein 2716 tnpR transposon Tn3 resolvase 2717 — hypothetical protein 2718 — hypothetical exported protein 2719 — conserved putative exported protein 2720 — conserved putative exported protein 2721 — conserved hypothetical protein 2722 — conserved hypothetical membrane protein 2723 — conserved hypothetical protein 2724 — hypothetical protein 2725 — hypothetical protein 2726 traC conserved putative antirestriction protein 2727 — possible type I restriction enzyme M subunit 2728 — hypothetical protein 2729 — conserved hypothetical protein 2730 — conserved hypothetical protein 2731 — conserved hypothetical protein 2732 — resolvase/integrase-like protein 2733 — hypothetical protein 2734 — hypothetical protein 2735 — conserved hypothetical protein 2736 — hypothetical protein 2737 — conserved hypothetical protein 2738 — putative site-specific recombinase 2739 thrC threonine synthase 2740 thrB homoserine kinase 2741 thrA aspartokinase/homoserine dehydrogenase 2742 — conserved hypothetical protein 2743 grk glycerate kinase 2744 — conserved hypothetical protein 2745 — conserved hypothetical protein 2746 — conserved hypothetical protein 2747 — conserved hypothetical protein 2748 — conserved hypothetical protein 2749 hitA iron-utilization periplasmic protein hFbpA 2750 hitB iron(III)-transport system permease protein 2751 hFbpB hitC iron-utilization ATP-binding protein hFbpC 2752 — putative D-alanyl-D-alanine carboxypeptidase 2753 dapE succinyl-diaminopimelate desuccinylase 2754 — conserved hypothetical protein 2755 — chaperone protein HtpG 2756 — conserved hypothetical NIF3-like protein 2757 hsdM1 putative type I restriction-modification system, 2758 methyltransferase subunit — predicted transcriptional regulator containing 2759 an HTH domain hsdS1 putative type I site-specific restriction-modification 2760 system, S subunit hsdR1 putative type I site-specific restriction-modification 2761 system, R subunit ffh Signal recognition particle protein 2762 corB putative Mg2+ and Co2+ transporter 2763 — conserved hypothetical protein 2764 — conserved hypothetical protein 2765 — conserved hypothetical protein 2766 serS Seryl-tRNA synthetase 2767 gst glutathione S-transferase 2768 — conserved hypothetical protein 2769 hemR hemin receptor 2770 — possible SAM-dependent methyltransferase 2771 mltA membrane-bound lytic murein transglycosylase A 2772 precursor — conserved hypothetical protein 2773 znuA high-affinity zinc uptake system protein ZnuA 2774 — conserved hypothetical protein 2775 mpl UDP-N-acetylmuramate:L-alanyl-gamma-D-glutamyl- 2776 meso-diaminopimelate ligase metC cystathionine beta-lyase 2777 tsaA probable peroxiredoxin 2778 pgsA CDP-diacylglycerol--glycerol-3-phosphate 3- 2779 phosphatidyltransferase ppa inorganic pyrophosphatase 2780 — conserved hypothetical protein 2781 udk uridine kinase 2782 dcd deoxycytidine triphosphate deaminase 2783 — conserved hypothetical protein 2784 — probable sugar efflux transporter 2785 engA GTP-binding protein EngA 2786 dnaQ DNA polymerase III, epsilon chain 2787 rnhA ribonuclease HI 2788 omP2 Outer membrane protein P2 precursor 2789 nagA N-acetylglucosamine-6-phosphate deacetylase 2790 nagB glucosamine-6-phosphate deaminase 2791 nanA N-acetylneuraminate lyase 2792 — putative HTH-type transcriptional regulator 2793 nanK putative N-acetylmannosamine kinase 2794 nanE putative N-acetylmannosamine-6-phosphate 2795 2-epimerase — putative sialic acid transporter, TRAP-type 2796 C4-dicarboxylate transport system, periplasmic component siaT putative sialic acid transporter, TRAP-type 2797 C4-dicarboxylate transport system, large permease component — conserved hypothetical protein 2798 — putative protein-S-isoprenylcysteine 2799 methyltransferase hflC HflC 2800 hflK HhflK 2801 — putative 4′-phosphopantetheinyl transferase 2802 dcuB2 anaerobic C4-dicarboxylate transporter DcuB 2803 acpP acyl carrier protein 2804 fabG 3-oxoacyl-[acyl-carrier protein] reductase 2805 fabD malonyl CoA-acyl carrier protein transacylase 2806 fabH 3-oxoacyl-[acyl-carrier-protein] synthase III 2807 rpmF 50S ribosomal protein L32 2808 — conserved hypothetical protein 2809 psd phosphatidylserine decarboxylase proenzyme 2810 gor glutathione reductase 2811 — conserved hypothetical lipoprotein 2812 nqrA Na(+)-translocating NADH-quinone reductase 2813 subunit A nqrB Na(+)-translocating NADH-quinone reductase 2814 subunit B nqrC Na(+)-translocating NADH-quinone reductase 2815 subunit C nqrD Na(+)-translocating NADH-quinone reductase 2816 subunit D nqrE Na(+)-translocating NADH-quinone reductase 2817 subunit E nqrF Na(+)-translocating NADH-quinone reductase 2818 subunit F apbE thiamine biosynthesis lipoprotein ApbE 2819 — conserved hypothetical protein 2820 trmU probable tRNA (5-methylaminomethyl-2- 2821 thiouridylate)-methyltransferase — conserved hypothetical protein 2822 rluD ribosomal large subunit pseudouridine synthase D 2823 — conserved hypothetical lipoprotein 2824 — conserved hypothetical protein 2825 pflA pyruvate formate-lyase 1 activating enzyme 2826 pflB formate acetyltransferase 2827 focA probable formate transporter 2828 — conserved hypothetical protein 2829 — putative Na+/alanine symporter 2830 — conserved hypothetical protein 2831 — putative HTH-type transcriptional regulator 2832 tatA Sec-independent protein translocase protein 2833 TatA/E tatB Sec-independent protein translocase protein 2834 TatB tatC Sec-independent protein translocase protein 2835 TatC gdhA NADP-specific glutamate dehydrogenase 2836 fur Ferric uptake regulation protein 2837 fldA flavodoxin 2838 seqA seqA 2839 — putative esterase/lipase 2840 menE O-succinylbenzoate--CoA ligase 2841 — putative small-conductance mechanosensitive 2842 channel aroC chorismate synthase 2843 mepA penicillin-insensitive murein endopeptidase 2844 — conserved hypothetical protein 2845 msbB lipid A biosynthesis (KDO)2-(lauroyl)-lipid IVA 2846 acyltransferase selD selenide, water dikinase 2847 rplsS 50S ribosomal protein L19 2848 trmD tRNA (guanine-N(1)-)-methyltransferase 2849 rimM 16S rRNA processing protein RimM 2850 rspP 30S ribosomal protein S16 2851 — conserved hypothetical protein 2852 nadN NAD nucleotidase 2853 aroK shikimate kinase 2854 aroB 3-dehydroquinate synthase 2855 dam DNA adenine methylase 2856 — conserved hypothetical protein 2857 pgpB phosphatidylglycerophosphatase B 2858 ribA GTP cyclohydrolase II 2859 — putative ABC-type oligopeptide transport system, 2860 periplasmic component — conserved hypothetical protein 2861 prlc oligopeptidase A 2862 Hsdm2 putative type I restriction-modification system 2863 methyltransferase subunit Hsds2 putative type I restriction-modification system 2864 specificity protein prrC putative anticodon nuclease 2865 — conserved hypothetical DNA binding protein 2866 Hsdr2 putative type I restriction-modification system 2867 — conserved hypothetical protein 2868 arcb aerobic respiration control sensor protein ArcB 2869 — predicted uracil-DNA glycosylase 2870 bira BirA bifunctional protein 2871 guab inosine-5′-monophosphate dehydrogenase 2872 guaa glutamine-hydrolyzing GMP synthase 2873 — conserved hypothetical protein 2874 — putative transcriptional regulator 2875 nhaa Na(+)/H(+) antiporter 1 2876 brnq branched-chain amino acid transport system 2877 carrier protein — conserved hypothetical protein 2878 — conserved hypothetical protein 2879 pnp polyribonucleotide nucleotidyltransferase 2880 nlpL lipoprotein NlpI 2881 dead Cold-shock DEAD-box protein A homolog 2882 — predicted soluble lytic transglycosylase fused 2883 to an ABC-type amino acid-binding protein — conserved hypothetical protein 2884 — conserved hypothetical protein 2885 — conserved hypothetical protein 2886 arsc putative arsenate reductase 2887 perm putative permease PerM homolog 2888 secf protein-export membrane protein SecF 2889 secd protein-export membrane protein SecD 2890 — conserved hypothetical preprotein translocase 2891 subunit YajC — predicted redox protein, regulator of disulfide 2892 bond formation — conserved hypothetical protein 2893 tgt queuine tRNA-ribosyltransferase 2894 quea S-adenosylmethionine:tRNA ribosyltransferase- 2895 isomerase — conserved hypothetical protein 2896 hap adhesion and penetration protein Hap 2897 uvra UvrABC system protein A 2898 ssb Single-strand binding protein 2899 tonb TonB 2900 exbd transport protein ExbD 2901 exbb transport protein ExbB 2902 bcp bacterioferritin comigratory protein 2903 dapa dihydrodipicolinate synthase 2904 — conserved hypothetical lipoprotein 2905 — conserved hypothetical protein 2906 lgtC UDP-galactose--lipooligosaccharide 2907 galactosyltransferase orfM predicted xanthosine triphosphate pyrophosphatase 2908 kdkA 3-deoxy-D-manno-octulosonic acid kinase 2909 opsX ADP-heptose--lipooligosaccharide 2910 heptosyltransferase I hxuC heme/hemopexin-binding protein C 2911 hxuB heme/hemopexin-binding protein B 2912 hxuA heme/hemopexin-binding protein A 2913 folB dihydroneopterin aldolase 2914 — conserved hypothetical protein 2915 narQ sensor protein NarQ 2916 murB UDP-N-acetylenolpyruvoylglucosamine reductase 2917 rpoH RNA polymerase sigma-32 factor 2918 djlA DnaJ-like protein DjlA 2919 pyrE orotate phosphoribosyltransferase 2920 rph ribonuclease PH 2921 gltX glutamyl-tRNA synthetase 2922 Lpt6 PE-tn-6--lipooligosaccharide 2923 phosphorylethanolamine transferase rbn tRNA processing ribonuclease BN 2924 — conserved hypothetical protein 2925 — conserved hypothetical protein 2926 udp uridine phosphorylase 2927 — conserved hypothetical metabolite transport 2928 protein — conserved hypothetical protein 2929 mend menaquinone biosynthesis protein MenD 2930 menf menaquinone-specific isochorismate synthase 2931 Aspc3 probable aspartate aminotransferase 2932 mtr tryptophan-specific transport protein 2933 sdaa L-serine dehydratase 2934 sdac Serine transporter 2935 — probable cation-transporting ATPase 2936 — probable heavy metal dependent transcriptional 2937 regulator metJ Met repressor 2938 rho transcription termination factor rho 2939 pilD putative type 4 prepilin-like protein specific 2940 leader peptidase pilC putative type IV pilin secretion protein 2941 pilB putative type IV pilin secretion protein 2942 pilA Type IV pilin subunit protein 2943 ampD ampD 2944 corC magnesium and cobalt efflux protein CorC 2945 cutE apolipoprotein N-acyltransferase 2946 — conserved hypothetical RNA methyltransferase 2947 — conserved hypothetical protein 2948 ruvX putative holliday junction resolvase 2949 — prophage CP4-57-like integrase 2950 — hypothetical protein 2951 — hypothetical protein 2952 — hypothetical protein 2953 — hypothetical protein 2954 — hypothetical protein 2955 — hypothetical protein 2956 proC pyrroline-5-carboxylate reductase 2957 hcaT probable 3-phenylpropionic acid transporter 2958 xerD Site-specific recombinase XerD 2959 — conserved hypothetical protein 2960 ruvB holliday junction DNA helicase RuvB 2961 ruvA holliday junction DNA helicase RuvA 2962 ruvC holliday junction DNA helicase RuvC 2963 — conserved hypothetical protein 2964 ntpA dATP pyrophosphohydrolase 2965 aspS Aspartyl-tRNA synthetase 2966 — conserved hypothetical protein 2967 — conserved hypothetical protein 2968 — conserved hypothetical protein 2969 — Predicted nucleic acid-binding domain, containsPIN 2970 domain gloA Lactoylglutathione lyase 2971 Rnt Ribonuclease T 2972 — conserved hypothetical protein 2973 — Predicted primosomal replication protein N 2974 Efp Elongation factor P 2975 — Predicted lysine 2,3-aminomutase 2976 oapA Opacity associated protein OapA 2977 oapB Opacity associated protein OapB 2978 recO DNA repair protein RecO 2979 rumA 23S rRNA (uracil-5--)methyltransferase RumA 2980 relA GTP pyrophosphokinase 2981 dgkA Diacylglycerol kinase 2982 Mog Molybdopterin biosynthesis mog protein 2983 glnB Nitrogen regulatory protein P-II 2984 — conserved hypothetical protein 2985 priA Prismosomal protein N 2986 trmB tRNA (guanine-N(7)-)-methyltransferase 2987 — conserved hypothetical protein 2988 napF Ferredoxin-type protein NapF 2989 napD NapD 2990 napA Periplasmic nitrate reductase 2991 napG Ferredoxin-type protein NapG 2992 napH Ferredoxin-type protein NapH 2993 napB Diheme cytochrome C NapB 2994 napC Cytochrome C-type protein NapC 2995 Adk Adenylate kinase 2996 Lic3c Putative integral membrane signal transducer protein 2997 galE UDP-glucose 4-epimerase 2998 Lic3A CMP-Neu5Ac--lipooligosaccharide alpha 2-3 2999 sialytransferase — Putative ABC-type nitrate/sulfonate/bicarbonate 3000 transport system, ATPase component — Putative ABC-type nitrate/sulfonate/bicarbonate 3001 transport system, permease component — Putative ABC-type nitrate/sulfonate/bicarbonate 3002 transport system, periplasmic component — Putative transcription activator 3003 hfeD Putative ABC-type chelated iron transport system, 3004 permease component hfeC Putative ABC-type chelated iron transport system, 3005 permease component hfeB Putative ABC-type chelated iron transport system, 3006 ATPase component hfeA Putative periplasmic chelated iron binding protein 3007 — Hypothetical protein 3008 pbpG Penicillin-binding protein 7 homolog precursos 3009 — Predicted Fe—S-cluster redox enzyme 3010 — Conserved hypothetical protein 3011 — Conserved hypothetical transcriptional regulator with 3012 an N-terminal xre-type HTH domain gcpE 4-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate 3013 synthase hisS Histidyl-tRNA synthetase 3014 — Conserved hypothetical protein 3015 — conserved hypothetical protein 3016 Fdx Feerdoxin, 2Fe—S 3017 hscA Chaperone protein HscA 3018 — Conserved hypothetical protein 3019 hscB Co-chaperone protein HscB 3020 — Conserved hypothetical protein 3021 nifU NifU-like protein 3022 nifS2 Cysteine sedulferase 3023 — Predicted transcriptional regulator 3024 — Hypothetical tRNA/rRNA methyltransferase 3025 Pal Outer membrane protein P6 precursor 3026 tolB TolB 3027 tolA TolA 3028 tolR TolR 3029 tolQ TolQ 3030 — Predicted thioestererase 3031 dinG Probable ATP-dependent helicase 3032 — Possible inactive homolog of metal-dependent 3033 proteases, putative molecular chaperone — possible starvation-inducible outer membrane 3034 lipoprotein lcfA Long chain fatty acid CoA ligase 3035 rnD Ribonuclease D 3036 — Conserved hypothetical acyltransferase 3037 — Predicted GTPase, probable translation factor 3038 Pth Peptidyl-tRNA hydrolase 3039 — Conserved hypothetical protein 3040 — Conserved hypothetical cupin superfamily 3041 metalloenzyme xseA Exodeoxyribonuclease VII large subunit 3042 adpP ADP-ribose pyrophoaphatase 3043 Icc Predicted phosphohydrolase 3044 — Conserved hypothetical protein 3045 ompP1 Outer membrane protein P1 precursor 3046 Ogt Methylated-DNA-protein-cysteine 3047 mutH DNA mismatch repair protein MutH 3048 mesJ Putative cell cycle protein MesJ 3049 accA Acetyl-coenzyme A carboxylase carboxyl transferase 3050 subunit alpha znuB High affinity zinc uptake system membrane protein 3051 ZnuB znuC High-affinity zinc uptake system ATP binding protein 3052 AnuC — Conserved hypothetical metalloprotease 3053 tyrR Transcriptional regulatory protein TyrR 3054 hfq host factor-I protein Hfq 3055 rluC ribosomal large subunit pseudouridine synthase C 3056 rne ribonuclease E 3057 — Conserved hypothetical protein 3058 thiM hydroxyethylthiazole kinase 3059 thiD phosphomethylpyrimidine kinase 3060 thiE thiamine-phosphate pyrophosphorylase 3061 — Conserved hypothetical metabolite transport 3062 — hypothetical protein 3063 — putative protease 3064 srmB ATP-dependent RNA helicase SrmB 3065 — predicted O-methyltransferase 3066 pssA CDP-diacylglycerol--serine O- 3067 phosphatidyltransferase fadR fatty acid metabolism regulator protein 3068 nhaB Na(+)/H(+) antiporter 2 3069 dsbB disulfide bond formation protein B 3070 glmS glucosamine--fructose-6-phosphate aminotransferase 3071 hupA DNA-binding protein HU 3072 — conserved hypothetical protein 3073 nudC NADH pyrophosphatase 3074 orfG conserved hypothetical 21.9 KD protein in locus 3075 involved in transformation comF competence protein F 3076 comE competence protein E 3077 comD competence protein D 3078 comC competence protein C 3079 comB competence protein B 3080 comA competence protein A 3081 mrcA penicillin-binding protein 1A 3082 — conserved hypothetical protein 3083 — conserved hypothetical protein 3084 recR recombination protein RecR 3085 topB DNa topoisomerase III 3086 secG protein-export membrane protein SecG 3087 fruA PTS system, fructose-specific IIBC component 3088 fruK 1-phosphofructokinase 3089 fruB PTS system, fructose-specific IIA/FPr component 3090 — conserved hypothetical protein 3091 vapD virulence-associated protein D 3092 vapX VapX 3093 — conserved hypothetical protein 3094 — conserved hypothetical protein 3095 — putative deoxyribonuclase 3096 holB DNA polymerase III, delta subunit 3097 — hypothetical protein 3098 lav autotransported protein Lav 3099 tmk thymidylate kinase 3100 — predicted periplasmic solute-binding protein 3101 surA survival protein SurA homolog 3102 pyrR PyrR bifunctional protein 3103 mazG predicted pyrophosphatase MazG 3104 — conserved hypothetical protein 3105 lon ATP-dependent protease La 3106 — predicted Fe—S oxidoreductase 3107 rpiA ribose 5-phosphate isomerase A 3108 serA D-3-phosphoglycerate dehydrogenase 3109 — predicted aminomethyltransferase related to GcvT 3110 — conserved hypothetical stress-induced protein 3111 hisG ATP phosphoribosyltransferase 3112 hisD histidinol dehydrogenase 3113 hisC hisitidinol-phosphate aminotransferase 1 3114 hisB histidine biosynthesis bifunctional protein HisB 3115 hisH imidazole glycerol phosphate synthase subunit HisH 3116 hisA 1-5-[methylideneamino] imidazole-4-carboxamine 3117 isomerase hisF imidazole glycerol phosphate synthase subunit HisF 3118 hisI histidine biosynthesis bifunctional protein hisIE 3119 — conserved hypothetical protein 3120 tyrP tyrosine-specific transportprotein 1 3121 atpC ATP synthase epsilon chain 3122 atpD ATP synthase beta chain 3123 atpG ATP synthase gamma chain 3124 atpA ATP synthase alpha chain 3125 atpH ATP synthase delta chain 3126 atpF ATP synthase B chain 3127 atpE ATP cynthase C chain 3128 atpB ATP synthase A chain 3129 — predicted F0F1-type ATP synthase subunit I 3130 gidB methyltransferase GidB 3131 — conserved hypothetical protein 3132 — predicted phosphatase/phosphohexomutase 3133 — predicted membrane protein 3134 luxS S-ribosylhomocysteinase 3135 aphA Class B acid phosphatase 3136 hslV ATP-dependent protease HslV 3137 hslU HslU, ATP-dependent chaperone of the HslUV 3138 protease ptoD2 spermidine/putrescine-binding periplasmic protein 2 3139 precursor ordL probable oxidoereductase OrdL 3140 rmuC DNA recombination protein RmuC 3141 rbsD ribose transport permease protein 3142 rbsA ribose transport ATP-binding protein 3143 rbsC ribose transport permease protein 3144 rbsB ribose-binding periplasmic protein 3145 rbsK ribokinase 3146 rbsR ribose operon repressor 3147 — predicted membrane protein 3148 menG S-adenosylmethionine:2-demtyhylmenaquinone 3149 methyltransferase menA 1,4-dihydroxy-2-naphthoate octaprenyltransferase 3150 — conserved hypothetical protein 3151 tehA tellurite resistance protein 3152 rpoC DNA-directed RNA polymerase beta′ chain 3153 rpoB DNA-directed RNA polymerase beta chain 3154 rplA 50S ribosomal protein L1 3155 rplK 50S ribosomal protein L11 3156 deoD purine nucleoside phosphorylase 3157 — predicted nucleoside permease 3158 — predicted pyruvate-formate lyase-activating enzyme 3159 — conserved hypothetical glycyl radical protein 3160 — predicted membrane protein 3161 waaQ ADP-heptose--lipooligosaccharide heptosyltransferase 3162 III fba frustose-biphosphate aldolase 3163 pgk phosphoglycerate kinase 3164 — probable rubonuclease I 3165 — putative ferredoxin-like protein 3166 tyrQ tyrosine-specific transport protein 2 3167 tdk thymidine kinase 3168 gcp probable O-sialoglycoprotein endopeptidase 3169 rpsU 30S ribosomal protein S21 3170 — DNA primase 3171 rpoD DNA polymerase sigma factor RpoD 3172 aspA aspartate ammonia-lyase 3173 ureH urease accessory protein UreH 3174 ureG urease accessory protein UreG 3175 ureF urease accessory protein UreF 3176 ureE urease accessory protein UreE 3177 ureC urease alpha subunit 3178 ureB urease beta subunit 3179 ureA urease gamma subunit 3180 groES 10 kDa chaperonin 3181 groEL 60 kDa chaperonin 3182 rplI 50S ribosomal protein L9 3183 rpsR 30S ribosomal protein S18 3184 priB primsomal replication protein N 3185 rpsF 30S ribosomal protein S6 3186 infA translation initiation factor IF-1 3187 ksgA dimethyladenosine transferase 3188 lic2A UDP-Gal--lipooligosaccharide galactosyltransferase 3189 apaH bis-tetraphosphatase, symmetrical 3190 — conserved hypothetical protein 3191 gnd 6-phosphogluconate dehydrogenase decarboxylating 3192 — conserved hypothetical protein 3193 — conserved hypothetical protein 3194 devB 6-phosphgluconolactonase 3195 zwf glucose-6-phosphate 1-dehydrogenase 3196 cysQ cysQ 3197 — conserved hypothetical protein 3198 — predicted membrane protein 3199 hslR heat shock protein 15 homolog 3200 asnC regulatory protein AsnC 3201 asnA aspartate--ammonia ligase 3202 — conserved hypothetical transposase-like protein 3203 — hypothetical protein 3204 — hypothetical protein 3205 — hypothetical protein 3206 gph phosphoglycolate phosphatase 3207 rpe ribulose-phosphate 3-epimerase 3208 gyrB DNA gyrase subunit B 3209 — predicted transcriptional accessory protein 3210 greB transcription elongation factor GreB 3211 — conserved hypothetical transcriptional regulator 3212 oxyR hydrogen peroxide-inducible genes activator 3213 pdgC peroxiredoxin•glutaredoxin 3214 slyX slyX 3215 fkby probably FKBP-type peptidyl-proyl cis-trans 3216 isomerase — conserved hypothetical protein 3217 — uncharacterized conserved protein involved in 3218 intracellular sulfur reduction — uncharacterized conserved protein involved in 3219 oxidation of intracellular sulfur — uncharacterized conserved protein involved in 3220 oxidation of intracellular sulfur tufB elongation factor Tu 3221 coaA pantothenate kinase 3222 rseB sigma-E factor regulatory protein RseB 3223 rseA sigma-E factor negative regulator protein homolog 3224 rpoE RNA polymerase sigma-E factor 3225 — conserved hypothetical protein 3226 mscL large-conductance mechanosensitive channel 3227 trkA Trk system potassium uptake protein TrkA 3228 sun SUN protein 3229 fmt methionyl-tRNA formyltransferase 3230 def peptide deformylase 3231 — hypothetical protein 3232 — hypothetical protein 3233 sxy DNA transformation protein TfoX 3234 recA RecA 3235 recX regulatory protein RecX 3236 crcB CrcB 3237 — predicted hydrolase of the HAD superfamily 3238 argF ornithine carbamoyltransferase, catabolic 3239 arcC carbamate kinase 3240 — predicted membrane protein 3241 hgpD pseudogene for hemoglobin-haptoglobin binding 3242 protein D pepE peptidase E 3243 — predicted C4-dicarboxylate transporter 3244 abgA aminobenzoyl-glutamate utilization protein A 3245 cpdB 2′,3′-cyclic-nucleotide 2′-phosphodiesterase 3246 — HTH-type transcriptional regulator 3247 — zinc transported ZitB 3248 gidA glucose inhibited division protein A 3249 rpsL 30S ribosoomal protein S12 3250 rpsG 30S ribosomal protein S7 3251 fusA elongation factor G 3252 tufB2 elongation factor Tu 3253 — predicted chloride channel protein 3254 — predicted chloride channel protein 3255 dusA tRNA-dihydrouridine synthase A 3256 — conserved hypothetical protein 3257 trpS tryptophanyl-tRNA synthetase 3258 — predicted protein involved in purine metabolism 3259 purB adenylosuccinate lyase 3260 rplJ 50S ribosomal protein L10 3261 rplL 50S ribosomal protein L7/L12 3262 glmU bifunctional GlmU protein 3263 — hypothetical protein 3264 pldB probable lysophospholipase L2 3265 asd aspartate-semialdehyde dehydrogenase 3266 — conserved hypothetical protein 3267 — predicted 2-methylthioadenine synthetase 3268 mdaB putative NADPH-quinone reductase, modulator of 3269 drug activity B rep ATP-dependent DNA helicase rep 3270 — predicted periplasmic lipoprotein 3271 kdtB phosphopantetheine adenylyltransferase 3272 kdtA 3-deoxy-D-manno-octulosonic acid transferase 3273 lgtF UDP-glucose--lipooligosaccharide glucosyltransferase 3274 tag DNA-3-methyladenine glycolase 3275 — hypothetical protein 3276 aroE shikimate 5-dehydrogenase 3277 predicted translation factor SUA5 3278 — Zn-finger domain associated with topoisomerase typr I 3279 — probable ABC transporter, ATP binding protein 3280 — putative HTH-type transcriptional regulator 3281 — conserved hypothetical protein 3282 hgpB hemoglobin-haptoglobin binding protein B 3283 — hypothetical ABC transporter, ATP-binding protein 3284 — probable ABC transporter, ATP binding protein 3285 — conserved hypothetical protein 3286 — conserved hypothetical protein 3287 — putative HTH-type transcriptional regulator 3288 glpX fructose-1,6-bisphosphatase class II GlpX 3289 — conserved hypothetical protein 3290 mioC MioC 3291 dtd D-tyrosyl-tRNA (Tyr) deacylase 3292 ispF 3-C-methyl-D-erythritol 2,4-cyclodiphosphate 3293 synthase ispD 2-C-methyl-D-erythritol 4-phosphate 3294 cytidylyltransferase ftsB cell division protein FtcB 3295 gpt xanthine-guanine phosphoribosyltransferase 3296 pepD aminoacyl-histidine dipeptidase 3297 xerC site-specific recombinase XerC 3298 — conserved hypothetical protein 3299 tpiA triosephosphate isomerase 3300 glpE thiosulfate sulfurtransferase GlpE 3301 — conserved hypothetical protein 3302 ilvY HTH-type transcriptional activator IlvY 3303 ilvC ketol-acid reductoisomerase 3304 glpC anaerobic glycerol-3-phosphate dehydrogenase 3305 subunit C glpB anaerobic glycerol-3-phosphate dehydrogenase 3306 subunit B glpA anaerobic glycerol-3-phosphate dehydrogenase 3307 subunit A glpT glycerol-3-phosphate transporter 3308 glpQ glycerophosphoryl diester phosphodiesterase precursor 3309 glpF glycerol uptake facilitator protein 3310 glpK glycerol kinase 3311 gpt2 xanthine-guanine phosphoribosyltransferase 3312 hel outer membrane protein P4, NADP phosphatase 3313 rluE ribosomal large subunit pseudouridine synthase E 3314 — conserved hypothetical protein 3315 — conserved hypothetical protein 3316 slyD FKBP-type peptidyl-prolyl cis-trans isomerase SlyD 3317 — conserved hypothetical protein 3318 truD tRNA pseudouridine synthase D 3319 surE acid phosphatase surE 3320 — conserved hypothetical protein 3321 — conserved hypothetical protein 3322 lppB outer membrane antigenic lipoprotein B 3323 tnaA tryptophanase 3324 tnaB tryptophan-specific transport protein 3325 mutS DNA mismatch repair protein MutS 3326 selA L-seryl-tRNA selenium transferase 3327 selB selenocysteine-specific elongation factor 3328 — hypothetical protein 3329 — conserved hypothetical protein 3330 — conserved hypothetical protein 3331 hgpC pseudogene for hemoglobin-haptoglobin utilization 3332 protein C tig trigger factor 3333 clpP ATP-dependent clp protease proteolytic subunit 3334 clpX ATP-dependent Clp protease ATP-binding subunit 3335 ClpX secE preprotein translocase SecE 3336 nusG transcription antitermination protein NusG 3337 vacJ VacJ lipoprotein 3338 — putative translation initiation inhibitor YjgF family 3339 htpX probable protease HtpX 3340 sirA SirA 3341 — conserved hypothetical protein 3342 trkH Trk system potassium uptake protein TrkH 3343 psiE PsiE 3344 hemY HemY 3345 hemX putative uroporphyrin-III C-methyltransferase 3346 cya adenylate cyclase 3347 gpsA glycerol-3-phosphate dehydrogenase 3348 cysE serine acetyltransferase 3349 — conserved hypothetical shikimate 5-dehydrogenase- 3350 like protein — possible di- and tricarboxylate transporter 3351 folD FolD bifunctional protein 3352 fucP L-fucose permease 3353 fucA L-fuculose phosphate aldolase 3354 fucU fucose operon protein FucU 3355 fucK L-fuculokinase 3356 fucI L-fucose isomerase 3357 fucR L-fucose operon activator 3358 hepA RNA polymerase associated protein homolog 3359 rluA ribosomal large subunit pseudouridine synthase A 3360 glpG GlpG 3361 glpR glycerol-3-phosphate regulon repressor 3362 metQ probable D-methionine-binding lipoprotein MetQ 3363 metI probable D-methionine transport system permease 3364 protein metN probable D-methionine transport ATP-binding protein 3365 — conserved hypothetical protein 3366 — conserved hypothetical protein 3367 — conserved hypothetical protein 3368 narP nitrate/nitrite response regulator protein 3369 lysA diaminopimelate decarboxylase 3370 — hypothetical protein 3371 cyaY CyaY 3372 recQ ATP-dependent DNA helicase RecQ 3373 proS prolyl-tRNA synthetase 3374 ostA organic solvent tolerance protein 3375 sufI SufI 3376 plsC 1-acyl-sn-glycerol-3-phosphate acyltransferase 3377 lpxH UDP-2,3-diacylglucosamine hydrolase 3378 — conserved hypothetical sodium dependent transporter 3379 ilvG acetolactate synthase isozyme II large subunit 3380 ilvD dihydroxy-acid dehydratase 3381 thd1 threonine dehydratase biosynthetic 3382 dnaE DNA polymerase III alpha subunit 3383 pgmB phosphoglucomutase 3384 secB protein-export protein SecB 3385 — predicted rhodanese-related sulfurtransferase 3386 dcuB anaerobic C4-dicarboxylate transporter DcuB 3387 ndhA NADH dehydrogenase 3388 plsB glycerol-3-phosphate acyltransferase 3389 lexA LexA repressor 3390 dapF diaminopimelate epimerase 3391 tpx probable thiol peroxidase 3392 purL phosphoribosylformylglycinamidine synthase 3393 lex2B UDP-glucose--lipooligosaccharide glucosyltransferase 3394 — conserved hypothetical protein 3395 — predicted membrane-bound metallopeptidase 3396 gpmA 2,3-bisphosphoglycerate-dependent phsphoglycerate 3397 mutase rpL31 50S ribosomal protein L31 3398 mutY A/G-specific adenine glycosylase 3399 — conserved hypothetical protein 3400 mltC membrane-bound lytic murein transglycolase C 3401 precursor — predicted diadenosine tetraphosphatase and related 3402 serine/threonine protein phosphatase nadR bifunctional protein NadR 3403 ribB 3,4-dihydroxy-2-butanone 4-phosphate synthase 3404 lpsA lipooligosaccharide glycosyl transferase 3405 — conserved hypothetical tRNA/rRNA methyltransferase 3406 — predicted N6-adenine-specific methylase 3407 FtsY cell division protein FtsY 3408 ftsE cell division ATP-binding protein EtsE 3409 ftsx cell division protein ftsx 3410 atoB acetyl-CoA acetyltransferase 3411 atoE short chain fatty acids transporter 3412 atoA acetate CoA-transferase beta subunit 3413 atoD acetate CoA-transferase alpha subunit 3414 — putative HTH-type transcriptional regulator 3415 rpsJ 30S ribosomal protein S10 3416 rplC 50S ribosomal protein L3 3417 rplD 50S ribosomal protein L4 3418 rplW 50S ribosomal protein L23 3419 rplB 50S ribosomal protein L2 3420 rpsS 30S ribosomal protein S19 3421 rplV 50S ribosomal protein L22 3422 rpsC 30S ribosomal protein S3 3423 rplP 50S ribosomal protein L16 3424 rpmC 50S ribosomal protein L29 3425 rpsQ 30S ribosomal protein S17 3426 — conserved hypothetical protein 3427 rplN 50S ribosomal protein L14 3428 rplX 50S ribosomal protein L24 3429 rplE 50S ribosomal protein L5 3430 rpsN 30S ribosomal protein S14 3431 rpsH 30S ribosomal protein S8 3432 rplF 50S ribosomal protein L6 3433 rplR 50S ribosomal protein L18 3434 rpsE 30S ribosomal protein S5 3435 rpmD 50S ribosomal protein L30 3436 rplO 50S ribosomal protein L15 3437 secY preprotein translocase SecY subunit 3438 rpsM 30S ribosomal protein S13 3439 rpsK 30S ribosomal protein S11 3440 rpsD 30S ribosomal protein S4 3441 rpoA DNA-directed RNA polymerase alpha chain 3442 rplQ 50S ribosomal protein L17 3443 — predicted cAMP-binding protein—catabolite gene 3444 activator and regulatory subunit of cAMP-dependent protein kinase — predicted permease 3445 dxr 1-deoxy-D-xylulose 5′phosphate reductoisomerase 3446 frr ribosome recycling factor 3447 pckA phosphoenolpyruvate carboxykinase 3448 hslO 33 kDa chaperonin 3449 argH argininosuccinate lyase 3450 galU UTP-glucose-1-phosphate uridylyltransferase 3451 csrA carbon storage regulator homolog 3452 alaS alanyl-tRNA synthetase 3453 uspA universal stress protein A 3454 pepP Xaa-Pro aminopeptidase 3455 — conserved hypothetical protein 3456 galM aldose 1-epimerase 3457 galK glactokinase 3458 galT galactose-1-phosphate uridylyltransferase 3459 galR HTH-type transcriptional regulator GalR 3460 mglB D-galactose-binding periplasmic protein precursos 3461 mglA galactoside transport ATP-binding protein MglA 3462 mglC galactoside transport system permease protein MglC 3463 — conserved hypothetical protein 3464 ispZ probable intracellular septation protein A 3465 — putative acyl-CoA thioester hydrolase 3466 — conserved hypothetical protein 3467 slt putative soluble lytic murein transglycosylase 3468 trpR Trp operon repressor 3469 mtgA monofuctional biosynthetic peptidoglycan 3470 transglycolase frdD fumarate reductase subunit D 3471 frdC fumarate reductase subunit C 3472 frdB fumarate reductase iron-sulfur protein 3473 frdA fumarate reductase flavoprotein subunit 3474 genX putative lysyl-tRNA synthetase 3475 cpxR transcriptional regulatory protein CpxR 3476 smpA small protein A 3477 ndpA nucleosid-associated protein NdpA 3478 — conserved hypothetical protein 3479 — predicted hydrolase of alkaline phosphatase 3480 superfamily — conserved hypothetical protein 3481 mobA probable molybdopterin-guanine dinucleotide 3482 biosyntheses protein A — conserved hypothetical protein 3483 dsbA thiol:disulfide interchange protein DsbA 3484 — conserved hypothetical protein 3485 trmA tRNA (Uracil-5)-methyltransferase 3486 — conserved hypothetical protein 3487 — predicted positive regulator of Sigma E 3488 mobB molybdopterin-guanine dinucleotide biosynthesis 3489 protein B — conserved hypothetical protein 3490 hbpA heme-binding protein A 3491 — putative heme iron utilization protein 3492 — conserved hypothetical protein 3493 polA DNA polymerase I 3494 — conserved hypothetical protein 3495 — predicted 5-formyltetrahydrofolate cyclo-ligase 3496 clpB ClpB 3497 — probable tRNA/rRNA methyltransferase 3498 vacB ribonuclease R 3499 — conserved hypothetical protein 3500 pdxH pyridoxamine 5′-phosphate oxidase 3501 typA GTP-binding protein TypA/BipA 3502 lic3A2 CMP-neu5Ac--lipooligosaccharide alpha 2-3 3503 sialyltransferase glnA glutamine synthetase 3504 rmlB dTDP-glucose 4,6-dehydratase 3505 pepB peptidase B 3506 ndk nucleoside diphosphate kinase 3507 — conserved hypothetical GTP-binding protein 3508 — conserved hypothetical transport protein 3509 rpmA 50S ribosomal protein L27 3510 rplU 50S ribosomal protein L21 3511 ispB octaprenyl-diphosphate synthase 3512 — conserved hypothetical protein 3513 — predicted Na+/alanine symporter 3514 arcA aerobic respiration control protein ArcA 3515 dsbD thiol:disulfide interchange protein DsbD 3516 — predicted membrane protein 3517 purH bifunctional purine biosynthesis protein PurH 3518 purD phosphoribosylamine--glycine ligase 3519 glyA serine hydroxymethyltransferase 3520 coaE dephospho-CoA kinase 3521 — conserved hypothetical zinc-binding protein 3522 rh1B ATP-dependent RNA helicase Rh1B 3523 — hypothetical transcriptional regulator 3524 — predicted membrane-fusion protein 3525 — predicted cation/multidrug efflux pump 3526 — predicted cell division protein 3527 emrB multidrug resistance protein 3528 emrA multidrug resistance protein A 3529 folA dihydrofolate reductase 3530 proB glutamate 5-kinase 3531 nudH probable nucleoside polyphosphate hydrolase 3532 — predicted permease 3533 lgt prolipoprotein diacylglyceryl transferase 3534 thyA thymidylate synthase 3535 — conserved hypothetical protein 3536 — conserved hypothetical protein 3537 — conserved hypothetical protein 3538 secA preprotein translocase SecA subunit 3539 mutT mutator protein MutT 3540 kefB glutathione-regulated potassium-efflux system protein 3541 — conserved hypothetical SAM-dependent 3542 methtransferase rpsB 30S ribosomal protein S2 3543 tsf elongation factor Ts 3544 lpxD UDP-3-O-[3-hydroxymyristoyl] glucosamine N- 3545 acyltransferase omp26 outer membrane protein 26 3546 — protective surface antigen D15 3547 — predicted membrane bound zinc matalloprotease with 3548 PDZ domain cdsA phosphaatidate cytidylyltransferase 3549 uppS undecaprenyl pyrophosphate synthetase 3550 leuS leucyl-tRNA synthetase 3551 — conserved predicted lipoprotein 3552 holA DNA polymerase III, delta subunit 3553 glyS glycyl-tRNA synthetase beta chain 3554 — hypothetical protein 3555 — conserved hypothetical protein 3556 — glutaredoxin-related protein 3557 glyQ glycyl-tRNA synthetase alpha chain 3558 hktE catalase 3559 — predicted glutathionylspermidine synthase 3560 — conserved hypothetical protein 3561 — conserved hypothetical protein 3562 eno enolase 3563 — conserved hypothetical protein 3564 nrfF formate-dependent nitrite reductase complex nrfFG 3565 subunit dsbE2 probable thiol:disulfide interchange protein DsbE 3566 nrfE cytochrome c-type biogenesis protein NrfE 3567 suhB inositol-1-monophosphatase 3568 — conserved hypothetical protein 3569 — predicted Type II secretory pathway, PulJ-like protein 3570 — conserved hypothetical protein 3571 — conserved hypothetical protein 3572 — exodeoxyribonuclease V gamma chain 3573 — predicted transcriptional regulator 3574 ribD riboflavin biosynthesis protein RibD 3575 degS protease DegS 3576 mutM formamidopyrimidine-DNA glycosylase 3577 ddc L-2,4-diaminobutyrate decarboxylase 3578 — predicted nucleic acid-binding protein, contains PIN 3579 domain — conserved hypothetical protein 3580 dat diaminobutyrate--2-oxoglutarate aminotransferase 3581 rpmG 50S ribosomal protein L33 3582 rpmB 50S ribosomal protein L28 3583 radC DNA repair protein Radc homolog 3584 dfp phosphopantothenoylcysteine 3585 synthetase/decarboxylase dut deoxyuridine 5′triphosphate nucleotidohydrolase 3586 ttk ttk 3587 — hypothetical protein 3588 crp catabolite gene activator 3589 — hypothetical RNA methyltransferase 3590 nagZ beta-hexosaminidase 3591 — predicted periplasmic lipoprotein 3592 — HIT-like protein 3593 ileS isoleucyl-tRNA synthetase 3594 ribF riboflavin biosynthesis protein RibF 3595 mviN putative virulence factor MviN 3596 rpsT 30S ribosomal protein S20 3597 — conserved hypothetical protein 3598 menB naphthoate synthase 3599 menC O-succinylbenzoate synthase 3600 aroQ 3-dehydroquinate dehydratase 3601 accB biotin carboxyl carrier protein of acetyl-CoA 3602 carboxylase accC biotin carboxylase 3603 — conserved hypothetical membrane protein 3604 panF sodium/pantothenase symporter 3605 — conserved hypothetical protein 3606 prmA ribosomal protein L11 methyltransferase 3607 dusB tRNA-dihydrouridine synthase B 3608 fis DNA-binding protein fis 3609 smpB SsrA-binding protein 3610 pfkA 6-phosphfructokinase 3611 — conserved hypothetical protein 3612 — conserved hypothetical protein 3613 smf smf 3614 leuA 2-isopropylmalate synthase 3615 leuB 3-isopropylmalate dehydrogenase 3616 leuC 3-isopropylmalate dehydratase large subunit 3617 leuD 3-isopropylmalate dehydratase small subunit 3618 igal IgA-specific serine endopeptidase 3619 recF DNA replication and repair protein RecF 3620 dnaN DNA polymerase III, beta chain 3621 dnaA chromosomal replication initiator protein DnaA 3622 tbp1 transferrin-binding protein 1 3623 tbp2 transferrin-binding protein 2 3624 — conserved hypothetical protein 3625 rpmH 50S ribosomal protain L34 3626 rnpA ribonuclease P protein component 3627 — conserved hypothetical protein 3628 yidC proprotein translocase subunit YidC 3629 trmE probable tRNA moficiation GTPase TrmE 3630 ppiD peptidyl-prolyl cis-trans isomerase D 3631 — predicted PR--lipooligosaccharide 3632 phosphorylethanolamine transferase lspA lipoprotein signal peptidase 3633 ispH 4-hydroxy-3-methylbut-2-enyl diphosphate reductase 3634 — conserved hypothetical protein 3635 tbpA thiamine-binding periplasmic protein 3636 thiP thiamine transport system permease protein 3637 thiQ thiamine transport ATP-binding protein 3638 bioB biotin synthase 3639 tktA transketolase 3640 serB phosphoserine phosphatase 3641 — conserved hypothetical protein 3642 corA magnesium and cobalt transport protein CorA 3643 — predicted integral membrane protein 3644 — predicted glutamine amidotransferase 3645 — hypothetical protein 3646 — predicted ATPase 3647 — hypothetical protein 3648 — predicted ferredoxin 3649 — conserved hypothetical protein 3650 dmsC anaerobic dimethyl sulfoxide reductase chain C 3651 dmsB anaerobic dimenthyl sulfoxide reductase chain B 3652 dmaA anaerobic dimethyl sulfoxide reductase chain A 3653 — conserved hypothetical protein 3654 — putative mercuric transport MerT homolog 3655 — predicted copper chaperone MerP homolog 3656 — conserved hypothetical ABC transporter 3657 — conserved hypothetical transcriptional regulator 3658 — conserved putative gamma-carboxymuconolactone 3659 decarboxylase subunit — conserved hypothetical protein 3660 res putative type III restriction-modification sustem 3661 HindVIP enzyme res rnhB ribonuclease HII 3662 lpxB lipid-A-disaccharide synthase 3663 lpxA acyl0[acyl-carrier-protein]--UDP-N- 3664 acetylglucosamine O-Acyltransferase fabZ (3R)-hydroxymyristoyl-[acyl carrier protein] 3665 dehydratase — predicted PR--lipooligosaccharide 3666 phosphorylethanolamine transferase pyrH uridylate kinase 3667 nrfD NrfD, formate-dependent nitrite reductase, membrane 3668 component nrfC NrfC, Fe—S-cluster-containing hydrogenase 3669 component 1 nrfB NrfB, cytochrome C-type protein 3670 nrfA cytochrome c552 3671 hrpA ATP-dependent helicase HrpA homolog 3672 — conserved putative small membrane protein 3673 — conserved putative membrane protein 3674 cyoB probable cyrochrome oxidase subunit II 3675 cyoA probable cytochrome oxidase dubunit I 3676 pyrG CTP synthase 3677 pnuC nictinamide riboside transporter 3678 — probable amino-acid ABC transporter ATP-binding 3679 protein — probable amino-acid ABC transporter permease 3680 protein — probable amino-acid ABC transporter binding protein 3681 murA UDP-N-acetylglucosamine a-carboxyvinyltransferase 3682 — predicted transcriptional regulator, BolA superfamily 3683 — predicted NTP binding protein, contains STAS domain 3684 — conserved ABC-type transport system protein 3685 — conserved ABC-type transport system protein, 3686 periplasmic component — conserved ABC-type transport system protein, 3687 permease component — conserved ABC-type transport system protein, ATPase 3688 component sodA superoxide dismutase [Mn] 3689 ccmA heme exporter protein A 3690 ccmB heme exporter protein B 3691 ccmC heme exporter protein C 3692 ccmD heme exporter protein D 3693 ccmE cytochrome c-type biogenesis protein CcmE 3694 ccmF cytochrome c-type biogenesis protein CcmF 3695 dsbE thiol:disulfide interchange protein DsbE 3696 — hypothetical protein 3697 — conserved hypothetical protein 3698 ligN DNA ligase 3699 zipA cell division protein ZipA 3700 cysZ CysZ 3701 cysK cysteine synthase 3702 rfaF ADP-heptose--lipooligosaccharide heptosyltransferase 3703 II xylR xylose operon refulatory protein 3704 — conserved hypothetical Na(+)/H(+) antiporter 3705 aspC2 putative aspartate aminotransferase 3706 xylA xylose isomerase 3707 xylB xylulose kinase 3708 rfaD ADP-L-glycero-D-manno-heptose-6-epimerase 3709 — thioredoxin-like protein 3710 deoC deoxyribose-phosphate aldolase 3711 comM competence protein ComM 3712 engB Probable GTP-binding protein EngB 3713 — D-xylose transport permease protein 3714 oppF oligopeptide transport ATP-binsing protein 3715 oppD oligopeptide transport ATP-binding protein 3716 oppC oligopeptide transport system permease protein 3717 oppB oligopeptide transport system permease protein 3718 oppA periplasmic oligopeptide-binding protein 3719 talB Transaldolase 3720 — carbon starvation protein, predicted membrane protein 3721 mraZ MraZ 3722 mraW predicted S-adenosylmethionine-dependent 3723 methyltransferase involved in cell envelope biogenesis ftsL cell division protein FtsL 3724 ftsI peptidoglycan synthetase FtsI 3725 murE UDP-N-acetylmuramoylalanyl-D-glutamate--2,6- 3726 diaminopimelate ligase murF UDP-N-acetylmuramoyl-tripeptide--D-alanyl-D- 3727 alanine ligase mraY phospho-N-acetylmuramoyl-pentapeptide-transferase 3728 murD UDP-N-acetylmuramoylaalanine--D-glutamate ligase 3729 ftsW cell division protein FtsW 3730 murG UDP-N-acetylglucosamine--N- 3731 acetylmuramylpyrophosphoryl-undecaprenol N- acetylglucosamine transferase murC UDP-N-acetylmuramate--L-alanine ligase 3732 ddlB D-alanine--D-alanine ligase 3733 ftsQ cell division protein FtsQ 3734 ftsA cell division protein FtsA 3735 ftsZ cell division protein FtsZ 3736 lpxC UDP-3-O-[3-hydroxymyristoyl] N-acetylglucosamine 3737 deacetylase pheA P-protein 3738 — predicted P-loop-containing kinase 3739 ptsN nitrogen regulatory IIA protein 3740 — probable ABC transporter ATP-binding protein 3741 — conserved hypothetical protein 3742 — conserved hypothetical protein 3743 — conserved hypothetical protein 3744 pmbA pmbA 3745 hpt hypoxanthine phosphoribosyltransferase 3746 — predicted Na+/dicarboxylate symporter 3747 nrdG anaerobic ribonucleoside-triphosphate reductase 3748 activating protein cydC transport ATP-binding protein CydC 3749 cydD transport ATP binding protein CydD 3750 trxB thioredoxin reductase 3751 — thioredoxin domain-containing protein 3752 hemH ferrochelatase 3753 — conserved hypothetical protein 3754 — conserved FAD/FMN-containing dehydrogenase 3755 ompP5 outer membrane protein P5 3756 — conserved glutaredoxin-related protein 3757 — histidinol-phosphate aminotransferase 2 3758 serC phosphoserine aminotransferase 3759 — conserved hypothetical protein 3760 — conserved hypothetical protein 3761 — conserved hypothetical protein 3762 trpG2 putative anthranilate synthase component II 3763 metK S-adenosylmethionine synthetase 3764 sprT SprT 3765 opa opacity protein 3766 — conserved hypothetical protein 3767 artM arginine transport system permease protein 3768 artQ arginine transport system permease protein 3769 artI arginine-binding periplasmic protein 3770 artP arginine transport ATP-binding protein 3771 gmhA phosphoheptose isomerase 3772 ligA DNA ligase 3773 dppF dipeptide transport ATP binding protein 3774 dppD dipeptide transport ATP binding protein 3775 dppC dipeptide transport system permease protein 3776 dppB dipeptide transport system permease protein 3777 uvrD DNA helicase II 3778 — predicted organic radical activating enzyme 3779 — predicted 6-pyruval-tetrahydropterin synthase 3780 — predicted PP-loop superfamily ATPase 3781 — conserved hypothetical protein 3782 ilvE branched chain amino acid amino transferase 3783 gcvA glycine cleavage system transcriptional activator 3784 — predicted SAM-dependent methyltransferase 3785 sucC succinyl-CoA synthetase beta chain 3786 sucD succinyl-CoA synthetase alpha chain 3787 — putative translation factor, Sua5 3788 rluB ribosomal large subunit pseudouridine synthase B 3789 cysB HTH-type transcriptional regulator CysB 3790 — conserved hypothetical adenine-specific methylase 3791 — conserved hypothetical protein 3792 pta phosphate acetyltransferase 3793 ackA acetate kinase 3794 — conserved hypothetical protein 3795 cvpA colicin C production protein 3796 — amidophosphoribosyltransferase 3797 sulA cell division inhibitor SulA 3798 argR argininr repressor 3799 mdh malate dehydrogenase 3800 lysS lysyl-tRNA synthetase 3801 prfB peptide chain release factor 2 3802 dsbC thiol:disulfide interchange protein DsbC 3803 recJ single stranded DNA specific exonuclease RecJ 3804 — conserved hypothetical protein 3805 mtnA MTA/SAH nucleosidase 3806 hup heme utilization protein 3807 — putative L-Lactate permease 3808 fbp frustose-1,6-bisphosphatase 3809 truA tRNA pseudouridine synthase A 3810 sapZ Predicted membrane protein 3811 sapF ABC-type transport system, ATPase component 3812 involved in antimicrobial peptide resistance sapD ABC-type transport system, ATP binding component, 3813 involved in antimicrobial peptide resistance sapC ABC-type transport system, permease protein, 3814 involved in antimicrobial peptide resistance sapB ABC-type transport system, permease protein, 3815 involved in antimicrobial peptide resistance sapA ABC-type transport system, periplasmic component, 3816 involved in antimicrobial peptide resistance — Predicted ATPase 3817 Ppc Phosphoenolpyruvate carboxylase 3818 purR HTH-type transcriptional repressor PurR 3819 dapD 2,3,4,5-tetrahydropyridine-2,6-dicarboxylate N- 3820 succinyltransferase PurA adenylosuccinate synthetase 3821 — predicted aspartokinase 3822 rplY 50S ribosomal protein L25 3823 — uncharacterized membrane-associate protein 3824 — conserved hypothetical protein 3825 — putative translation initiation inhibitor, YjgF family 3826 — conserved hypothetical protein 3827 — conserved hypothetical protein 3828 — HTH-type trancriptional regulator 3829 — putative ABC-type Co2+ transport system, 3830 periplasmic component — conserved hypothetical protein 3831 cbiM predicted ABC-type cobalt transport system, permease 3832 component — predicted cobalt transport protein 3833 cbiO predicted ABC-type cobalt transport system, ATPase 3834 component aspC aspartate aminotransferase 3835 purK phosphoribosylaminoimidazole carboxylase ATPase 3836 subunit purE phosphoribosylaminoimidazole carboxylase catalytic 3837 subunit hicA HicA 3838 hicB HicB 3839 pepN aminopeptidase N 3840 ribE riboflavin synthase alpha chain 3841 norM probable multidrug resistance protein NorM 3842 sfsA sugar fermentation stimulation protein 3843 tyrS tyrosyl-tRNA synthase 3844 prsA ribose phosphate pyrophosphokinase 3845 ispE 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase 3846 lolB outer-membrane lipoprotein LolB 3847 cca tRNA nucleotidyltransferase 3848 — conserved hypothetical protein 3849 — putative phosphate permease 3850 — conserved hypothetical phosphate transport regulator 3851 — predicted membrane protein 3852 — conserved hypothetical protein 3853 — conserved hypothetical protein 3854 — conserved hypothetical protein 3855 — hypothetical protein 3856 — hypothetical protein 3857 — hypothetical protein 3858 hmw2C HMW2C, putative glycosyltransferase involved in 3859 glycosylation of HMW1A and HMW2A hmw2B HMW2B, OMP-85-like protein required for HMW1A 3860 and HMW2A secretion —hmw2A HMW2A, high molecular weight adhesin 2 3861 — conserved hypothetical protein 3862 radA DNA repair protein RadA homolog 3863 lrp leucine-responsive regulatory protein 3864 ftsK DNa translocase FtsK 3865 lolA outer-membrane lipoproteins carrier protein precursor 3866 — predicted ATPase related to the helicase subunit of the 3867 holliday junction resolvase — hypothetical protein 3868 — modification methylase BepI-like 3869 aroA 3-phosphoshikimate 1-carboxyvinyltransferase 3870 purU formyltetrahydrofolate deformylase 3871 hns DNA-binding protein H-NS homolog 3872 — predicted Na+/H+ antiporter 3873 — hypothetical protein 3874 ilvI acetolactate synthase large subunit 3875 ilvH acetolactate synthase small subunit 3876 argS arginyl-tRNA synthetase 3877 — conserved hypothetical protein 3878 — hypothetical lipoprotein 3879 pcp outer-membrane lipoprotein PCP precursor 3880 lgtD UDP-0glcNAc-lipooligosaccharide N- 3881 acetylglucosamine glycosyltransferase pgi glucose-6-phosphate isomerase 3882 alr alanine racemase 3883 dnaB replicative DNA helicase 3884 pykA pyruvate kinase 3885 — prophage CP4-57-like integrase 3886 — hypothetical protein 3887 — hypothetical protein 3888 — hypothetical protein 3889 — hypothetical protein 3890 — hypothetical protein 3891 — modification methylase Bsp6I-like 3892 rdgC recombination associated protein 3893 — hypothetical protein 3894 ssb3 single strand binding protein 3895 — hypothetical protein 3896 — predicted recombinational DNA repair protein, RecE 3897 pathway — hypothetical protein 3898 — hypothetical protein 3899 — modification methylase DpnIIB-like 3900 — hypothetical protein 3901 — hypothetical protein 3902 — hypothetical protein 3903 — hypothetical protein 3904 — hypothetical protein 3905 — hypothetical protein 3906 — hypothetical protein 3907 — hypothetical protein 3908 — hypothetical protein 3909 — hypothetical protein 3910 — hypothetical protein 3911 — hypothetical protein 3912 — hypothetical protein 3913 — hypothetical protein 3914 — hypothetical protein 3915 — hypothetical protein 3916 — hypothetical protein 3917 — hypothetical protein 3918 — hypothetical protein 3919 — hypothetical protein 3920 — hypothetical protein 3921 — hypothetical protein 3922 — hypothetical protein 3923 — predicted DNA modification methylase 3924 — hypothetical protein 3925 — hypothetical protein 3926 — predicted phage terminase large subunit 3927 — hypothetical protein 3928 — uncharacterized protein, homolog of phage Mu protein 3929 gp30 — hypothetical protein 3930 — hypothetical protein 3931 — hypothetical protein 3932 — hypothetical protein 3933 — hypothetical protein 3934 — hypothetical protein 3935 — hypothetical protein 3936 — hypothetical protein 3937 — hypothetical protein 3938 — hypothetical protein 3939 — hypothetical protein 3940 — hypothetical protein 3941 — predicted phage-related minor tail protein 3942 — hypothetical protein 3943 — hypothetical protein 3944 — hypothetical protein 3945 — hypothetical protein 3946 — hypothetical protein 3947 — hypothetical protein 3948 — hypothetical protein 3949 — hypothetical protein 3950 — probable tail fiber protein 3951 — hypothetical protein 3952 — hypothetical protein 3953 — conserved hypothetical protein 3954 — hypothetical protein 3955 — hypothetical protein 3956 — mu-like prophage protein gp29 3957 — conserved hypothetical protein 3958 — hypothetical protein 3959 — conserved hypothetical protein 3960 — hypothetical protein 3961 — hypothetical protein 3962 — hypothetical protein 3963 — hypothetical protein 3964 — hypothetical protein 3965 — hypothetical protein 3966 — hypothetical protein 3967 prfA peptide chain release factor 1 3968 — conserved hypothetical protein 3969 hemK HemK 3970 — conserved hypothetical protein 3971 kdsA 2-dehyrdro-3-deoxyphosphooctonate aldolase 3972 — putative 2-hydroxyacid dehydrogenase 3973 lolC lipoprotein releasing system transmembrane protein 3974 bioA adenosylmethionine-8-amino-7-oxononanoate 3975 aminotransferase bioF 8-amino-7-oxononanoate synthase 3976 — conserved hypothetical protein 3977 bioC putative biotin synthesis protein BioC 3978 bioD-B probable dethiobiotin synthetase 2 3979 lolD lipoprotein releasing system ATP-binding protein 3980 LolD lolE ABC-type transport system, involved in lipoprotein 3981 release, permease component aroG phospho-2-dehyrdro-3-deoxyheptonate aldolase 3982 impA impA 3983 — predicted Na+/serine symporter 3984 — putative NAD(P)H oxidoreductase 3985 — predicted component of anaerobic dehydrogenases 3986 — predicted nitroreductase 3987 sppA protease IV 3988 licD phosphorylcholine transferase 3989 licC LicC 3990 licB LicB 3991 licA LicA 3992 — predicted glycine/D-amino acid oxidases, deaminating 3993 fabB 3-oxacyl-[acyl-carrier-protein] synthase I 3994 rrxA glutaredoxin 3995 rimK probable ribosomal protein S6 modification protein 3996 gltS sodium/glutamate symport carrier protein 3997 parC topoisomerase IV subunit A 3998 parE topoisomerase IV subunit B 3999 htrB lipid A biosynthesis lauroyl acyltransferase 4000 rfaE ADP-heptose synthase 4001 — hypothetical protein 4002 uupA2 ABC transporter ATP-binding protein 4003 uppB ABC transporter ATP-binding protein 4004 — putative carbonic anhydrase 4005 asnS asparaginyl-tRNA synthetase 4006 ribH 6,7-dimethyl-8-ribityllumazine synthase 4007 nusB N utilization substance protein B 4008 thiL thiamine-monophosphate kinase 4009 pgpA phosphatidylglycerophosphatase A 4010 — predicted threonine efflux protein 4011 dapB dihydrodipicolinate reductase 4012 — conserved hypothetical ferredoxin-like protein 4013 — conserved hypothetical protein 4014 pheS ohenylalanyl-tRNA synthetase alpha chain 4015 pheT ohenylalanyl-tRNA synthetase beta chain 4016 himA integration host factor alpha-subunit 4017 — conserved hypothetical lipoprotein 4018 — hypothetical protein 4019 — putative 5′(3′)-deoxyribonucleotidase 4020 — NAD-dependent deacetylase 4021 — hypothetical protein 4022 ftsK2 DNA translocase ftsK 4023 — NAD-dependent deacetylase sirtuin 5 4024 — hypothetical protein 4025 — hypothetical protein 4026 — hypothetical protein 4027 — predicted serine/threonine protein phosphatase family 4028 protein — predicted arylsulfatase A-like enzyme 4029 — predicted enzyme related to aldose 1-epimerase 4030 infC translation initiation factor IF-3 4031 rpmI 50S ribosomal protein L35 4032 rplT 50S ribosomal protein L20 4033 recB exodeoxyribonuclease V beta chain 4034 recD exodeoxyribonuclease V alpha chain 4035 — conserved hypothetical protein 4036 lonB putative protease Lahomolog, predicted ATP- 4037 dependent protease fabA 3-hydroxydecanoyl-[acyl-carrier-protein] dehydratase 4038 — conserved hypothetical protein 4039 — conserved hypothetical protein 4040 rpsO 30S ribosomal protein S15 4041 — conserved hypothetical transposase-like protein 4042 — conserved hypothetical protein 4043 dacB penicillin-binding protein 4 precursor 4044 greA transcription elongation factor GreA 4045 — predicted RNA-binding protein containing KH 4046 domain, possible ribosomal protein ftsJ ribosomal RNA large subunit methyltransferase J 4047 FTSh cell division protein FtsH homolog 1 4048 folP dihydropteroate synthase 4049 mrsA predicted phosphomannomutase 4050 sixA phosphohistidine phosphatase SixA homolog 4051 — conserved hypothetical protein 4052 — conserved hypothetical protein 4053 — hypothetical protein 4054 — hypothetical protein 4055 — possible RNA polymerase sigma factor 24 4056 — hypothetical protein 4057 — hypothetical protein 4058 msaB peptide methionine sulfoxide reductase MsrA/MsrB 4059 — conserved hypothetical cytochrome c-type biogenesis 4060 protein — conserved hypothetical protein 4061 — conserved hypothetical protein 4062 moeB molybdopterin biosynthesis protein MoeB 4063 moeA molybdopterin biosynthesis protein MoeA 4064 folE GTP cyclohydrolase I 4065 — conserved hypothetical protein 4066 bioD-A probable dethiobiotin synthetase 1 4067 metF 5,10-methylenetetrahydrofolate reductase 4068 rplM 50S ribosomal protein L13 4069 rpsI 30S ribosomal protein S9 4070 sspA stringent starvation protein A 4071 sspB stringent starvation protein B 4072 dxs 1-deoxy-D-xylulose 5-phosphate synthase 4073 ispA geranyltranstransferase 4074 xseB exodeoxyribonuclease VII small subunit 4075 thiI predicted thiamine biosynthesis ATP pyrophosphatase 4076 — conserved hypothetical protein 4077 truC tRNA pseudouridine synthase C 4078 — conserved hypothetical protein 4079 cspD cold shock-like protein CspD 4080 — conserved hypothetical protein 4081 usg predicted aspartate-semialdehyde dehydrogenase 4082 trpA tryptophan synthase alpha chain 4083 trpB tryptophan synthase beta chain 4084 — conserved hypothetical oxidoreductase 4085 purM phosphoribosylformylglycinamidine cyclo-ligase 4086 purN phosphoribosylglycinamide formyltransferase 4087 — predicted ABC-type transport system protein, 4088 periplasmic component uspE universal stress protein E 4089 fnr fumarate and nitrate reduction regulatory protein 4090 — putative integrase/recombinase 4091 — conserved hypothetical protein 4092 — predicted phage anti-repressor protein 4093 — hypothetical protein 4094 — hypothetical protein 4095 — hypothetical protein 4096 — hypothetical protein 4097 — hypothetical protein 4098 — hypothetical protein 4099 — predicted transcriptional regulator 4100 — hypothetical protein 4101 — hypothetical protein 4102 — hypothetical protein 4103 — hypothetical protein 4104 — hypothetical protein 4105 — hypothetical protein 4106 ninB putative recombination protein NinB 4107 ninG putative recombination protein NinG homolog 4108 — hypothetical protein 4109 — conserved hypothetical protein 4110 — hypothetical protein 4111 — conserved hypothetical protein 4112 — hypothetical protein 4113 — conserved hypothetical protein 4114 — conserved hypothetical protein 4115 — hypothetical protein 4116 — conserved hypothetical protein 4117 — hypothetical protein 4118 — conserved hypothetical protein 4119 — predicted phage terminase large subunit 4120 — conserved hypothetical protein 4121 — uncharacterized protein, homolog of phage Mu protein 4122 gp30 — conserved hypothetical protein 4123 pyrD dihyrdoorotate dehydrogenase 4124 trpH TrpH 4125 — conserved hypothetical protein 4126 fumC fumurate hydratase class II 4127 — putative glycosyl transferase, glycosyl transferase 4128 family 8 protein holC DNA polymerase III, chi subunit 4129 — conserved hypothetical protein 4130 — conserved hypothetical protein 4131 — hypothetical protein 4132 — conserved hypothetical protein 4133 — hypothetical protein 4134 valS valyl-tRNA synthetase 4135 — hypothetical protein 4136 trpC tryptophan biosynthesis protein trpCF 4137 trpD anthranilate phosphoribosyltransferase 4138 — conserved hypothetical protein 4139 trpG anthranilate synthase component II 4140 trpE anthranilate synthase component I 4141 — pseudogene for conserved hypothetical 4142 glycosyltransferase ftnB ferritin-like protein 2 4143 ftnA ferritin-like protein 1 4144 pstS phosphate-binding periplasmic protein precursor PstS 4145 pstC phosphate transport system permease protein PstC 4146 pstA phoaphate transport system permease proteain PstA 4147 pstB phosphate import ATP-binding protein PstB 4148 phoB phosphate regulon transcriptional regulatory protein 4149 PhoB phoR phosphate regulon sensor protein PhoR 4150 sbcB exodeoxyribonuclease I 4151 — conserved hypothetical protein 4152 — conserved hypothetical protein 4153 — cell division protein MukB 4154 — cell division protein MukE 4155 haeIIR type II restriction exzyme HaeII 4156 haeIIM modification methylase HaeII 4157 mukF MukF homolog 4158 — predicted ATPase of the PP-loop superfamily 4159 implicated in cell cycle control — predicted dissimilatory sulfite reductase, 4160 desulfoviridin, gamma subunit mop probable molybdenum-pterin binding protein 4161 pqqL probable zinc protease 4162 thrS threonyl-tRNA synthetase 4163 acpD probable acyl carrier protein phosphodiesterase 4164 topA DNA topoisomerase I 4165 — putative HTH-type transcriptional regulator 4166 pntB NAD(P) transhydrogenase subunit beta 4167 pntA NAD(P) transhydrogenase subunit alpha 4168 glgP glycogen phosphorylase 4169 glgA glycogen synthase 4170 glgC glucose-1-phosphate adenylyltransferase 4171 glgX glycogen operon protein GlgX 4172 glgB 1,4-alpha-glucan branching enzyme 4173 malQ 4-alpha-glucanotransferase 4174 — conserved hypothetical protein 4175 glnS glutaminyl-tRNA synthetase 4176 cafA ribonuclease G 4177 putP sodium/proline symporter 4178 — conserved hypothetical protein 4179 cdd cytiding deaminase 4180 — conserved hypothetical DNA-binding ferritin-like 4181 protein pepT peptidase T 4182 potA spermidine/putrescine transport ATP-binding protein 4183 potB spermidine/putrescine transport permease protein 4184 potC spermidine/putrescine transport system permease 4185 protein potD1 spermidine/putrescine-binding periplasmic protein 1 4186 precursor uupA ABC transporter ATP-binding protein 4187 — deoxyguanosinetriphosphate triphosphohydrolase-like 4188 protein — putative effector of murein hydrolase 4189 — putative effector of murein hydrolase 4190 — predicted micrococcal nuclease-like protein 4191 nifS predicted selenocysteine lyase 4192 — predicted SufE protein probably involved in Fe—S 4193 center assembly — Zn-ribbon-containing, possible nucleic-acid binding 4194 protein — predicted enzyme related to GTP cyclohydrolase I 4195 tyrA T-protein 4196 truB tRNA pseudouridine synthase B 4197 rbfA ribosome-binding factor A 4198 hsdM3 putative type I restriction enzyme HindVIIP M protein 4199 hsdS3 putative type I restriction enzyme HindVIIP 4200 specificity protein — hypothetical protein 4201 hsdR3 putative type I restriction enzyme HindVIIP R protein 4202 infB translation initiation factor IF-2 4203 nusA transcriptional elongation protein NusA 4204 — conserved hypothetical protein 4205 — hypothetical protein 4206 — hypothetical protein 4207 — hypothetical protein 4208 — hypothetical protein 4209 — hypothetical protein 4210 — hypothetical protein 4211 — hypothetical protein 4212 — probable tail fiber protein 4213 — predicted bacteriophage P2-related tail formation 4214 protein gpI — predicted phage-related baseplate assembly protein 4215 — predicted baseplate assembly protein W 4216 — predicted phage P2-like baseplate assembly protein 4217 — hypothetical protein 4218 — hypothetical protein 4219 — hypothetical protein 4220 — hypothetical protein 4221 — predicted phage-related tail protein 4222 — hypothetical protein 4223 — hypothetical protein 4224 — hypothetical protein 4225 — hypothetical protein 4226 — probable bacteriophage tail completion protein gpS 4227 homolog — probable bacteriophage tail completion protein gpR 4228 homolog — hypothetical protein 4229 — hypothetical protein 4230 — DnaK suppressor protein, bacteriophage PSP3 gp34 4231 homolog — hypothetical protein 4232 — predicted phage-related lysozyme 4233 — hypothetical protein 4234 — hypothetical protein 4235 — hypothetical protein 4236 — predicted terminase, endonuclease subunit 4237 — predicted major capsid protein 4238 — predicted capsid scaffolding protein 4239 — terminase, ATPase subunit 4240 — predicted portal vertex protein 4241 siaB acylneuraminate cytidylyltransferase 4242 — putative NAD(P)H nitroreductase 4243 mrp Mrp 4244 metG methionyl-tRNA synthetase 4245 tehB tellurite resistance protein 4246 gloB probable hydroxyacylglutathione hydrolase 4247 — conserved hypothetical protein 4248 — conserved hypothetical protein 4249 — conserved hypothetical protein 4250 gyrA DNA gyrase subunt A 4251 metX homoserine O-acetyltransferase 4252 sanA SanA 4253 folC folypolyglutamate synthase 4254 accD acethy-coenzyme A carboxylase carboxyl transferase 4255 subunit beta htoA probable periplasmic serine protease do/HhoA-like 4256 precursor mfd transcription-repair coupling factor 4257 — conserved hypothetical protein 4258 — predicted P-loop ATPase fused to an acetyltransferase 4259 — conserved hypothetical protein 4260 — ABC transported ATP-binding protein 4261 — predicted plasmid maintenance system antidote protein 4262 — predicted plasmid maintenance system killer protein 4263 — predicted ABC-type transport system, periplasmic 4264 component — predicted ABC-type transport system, permease 4265 component uvrB UvrABC system protein B 4266 — predicted phosphoglycerol transferase-like protein 4267 mao2 NADP-dependent malic enzyme 4268 — possible polysaccharide biosynthesis protein 4269 rsuA ribosomal small subunit pseodouridine synthase A 4270 bcr bicyclomycin resistance protein 4271 — conserved hypothetical protein 4272 — predicted membrane protein 4273 proA gamma-glutamyl phosphate reductase 4274 dnaJ chaperone protein DnaJ 4275 dnaK chaperone protein DnaK 4276 — conserved hypothetical protein 4277 mgsA methylglyoxal synthase 4278 aceE pyruvate dehydrogenase E1 component 4279 aceF dihydrolipoamide acetyltransferase component of 4280 pyruvate dehydrogenase complex lpdA dihydrolipoamide dehudrogenase 4281 — hypothetical protein 4282 apt adenine phosphoribosyltransferase 4283 dnzX DNA polymerase III subunit gamma/tau 4284 upp uracil phosphoribosyltransferase 4285 uraA probable uracil permease 4286 — predicted ATPase involved in DNA replication 4287 initiation — predicted translation initiation factor 1-like proterin 4288 pyrF orotidine 5′-phosphate decarboxylase 4289 — predicted N-acetylglucosaminyl transferase 4290 — predicted membrane protein 4291 ihfB integration host factor beta subunit 4292 rpsA 30S ribosomal protein S1 4293 cmk ctidylate kinase 4294 — conserved hypothetical pyridoxine biosynthesis 4295 enzyme — predicted glutamine amidotransferase involved in 4296 pyridoxine biosynthesis dld D-lactate dehydrogenase 4297 — conserved hypothetical protein 4298 nlpC probable lipoprotein NlpC 4299 tldD TldD 4300 — predicted methyltransferase 4301 — putative lipoprotein 4302 — predicted endonuclease distantly related to archael 4303 holliday junction resolvase — predicted phosphoheptose isomerase 4304 — predicted periplasmic or secreted lipoprotein 4305 nrdA ribonucleoside-diphosphate reductase alpha chain 4306 nrdB ribonucleoside-diphosphate reductase beta chain 4307 sucB dihydrolipoamide succinyltransferase component of 4308 2-oxoglutarate dehydrogenase complex sucA 2-oxoglutarate dehydrogenase E1 component 4309 — predicted Zn-dependent hydrolase-like protein, 4310 including glyoxylases — conserved putative deoxyribonuclease 4311 — conserved hypothetical protein 4312 — conserved hypothetical protein 4313 prc tail-specific protease precursor 4314 proQ predicted activator of osmoprotectant transporter 4315 PropP — paraquat-inducible protein A-like protein 4316 — paraquat-inducible protein B-like protein 4317 moaE molybdopterin converting factor subunit 2 4318 moaD molybdopterin converting factor subunit 1 4319 moaC molybdenum cofactor biosynthesis protein C 4320 moaA molybdenum cofactor biosynthesis protein A 4321 — predicted regulator of cell morphogenesis and NO 4322 signaling predicted regulator of cell morphogenesis and NO 4323 signaling — probable phosphosugar isomerase Hi1678 4324 yrbI 3-deoxy-D-manno-octulosonate 8-phosphate 4325 phosphatase hmw1A HMW1A, high molecular weight adhesin 1 4326 hmw1B HMW1B, OMP-85-like protein required for secretion 4327 of HMW1A and HMW2A hmw1C HMW1C, putative glycosyltransferase involved in 4328 glycosylation of HMW1A and HWM2A — predicted membrane protein 4329 — conserved hypothetical protein 4330 sohB possible protease SohB 4331 rnfA predicted NADH:ubiquinone oxidoreductase, subunit 4332 RnfA rnfB predicted NADH:ubiquinone oxidoreductase, subunit 4333 RnfB rnfC predicted NADH:ubiquinone oxidoreductase, subunit 4334 RnfC rnfD predicted NADH:ubiquinone oxidoreductase, subunit 4335 RnfD rnfG predicted NADH:ubiquinone oxidoreductase, subunit 4336 RnfG — predicted NADH:ubiquinone oxidoreductase, subunit 4337 RnfE nth endonuclease III 4338 — predicted Na+-dependent transporters of the SNF 4339 family modC molybdenum import ATP-binding protein 4340 modB molybdenum transport system permease protein 4341 modA molybdate-binding periplasmic protein 4342 mode Transcriptional regulator ModE 4343 lsgF Putative UDP-galactose-lipooligosaccharide 4344 galactosyltransferase lsgE Putative UDP-galactose-lipooligosaccharide 4345 galactosyltransferase lsgD Putative UDP-glcNAc-lipooligosaccharide N- 4346 acetylglucosaminyl glycosyltransferase lsgC Putative UDP-galactose--lipooligosaccharide 4347 galactosyltransferase lsgB CMP-N-acetylneuraminate-beta-galactosamide-alpha- 4348 2,3-sialytransferase lsgA putative lipooligosaccharide flippase 4349 — conserved hypothetical protein 4350 — predicted permease 4351 — predicted permease 4352 pepA cytosol aminopeptidase 4353 — predicted choline-glycine betaine transporter 4354 qseC sensor protein QseC 4355 qseB transcriptional regulatory protein QseB 4356 — conserved hypothetical protein 4357 crr PTS system, glucose-specific IIA component 4358 ptsI Phosphoenolpyruvate-protein phosphotransferase 4359 ptsH Phosphocarrier protein HPr 4360 — probable GTPase 4361 orn oligoribonuclease 4362 wecA undecaprenyl phosphate 4363 glnD [protein-PII] uridylyltransferase 4364 map methionine aminopeptidase 4365 — conserved hypothetical protein 4366 — conserved hypothetical protein 4367 mrcB penicillin-binding protein 1B 4368 purC phosphoribosylaminoimidazole-succinocarboxamine 4369 synthase argG argininosuccinate synthase 4370 — Mn2+ and Fe2+ transporter of the NRAMP family 4371 — conserved hypothetical protein 4372 — predicted allophanate hydrolase subunit 2 4373 — predicted allophanate hydrolase subunit 1 4374 rnb exoribonuclease II 4375 fabI enoyl-[acyl-carrier-protein] reductase [NADH] 4376 prfC peptide chain release factor 3 4377 — conserved hypothetical protein 4378 — predicted branched chain amino acid permease 4379 — predicted branched chain amino acid permease 4380 metR HTH-type transcriptional regulator MetR 4381 lldD L-lactate dehydrogenase 4382 murI glutamate racemase 4383 recG ATP-dependent DNA helicase 4384 spoT guanosine-3′,5′-bis 3′pyrophosphohydrolase 4385 rpoZ DNA directed RNA polymerase omega chain 4386 gmk guanylate kinase 4387

Example 10 Comparison of the Genomes of NTHi, Strain 86-028NP and H. influennzae, Strain RD

The genomic sequence of strain 86-028NP contains 1,913,428 bp. This is approximately 4 percent larger than the strain Rd genome (1,830,137 bp) (Fleischmann et al., Science 269:496-512, 1995). There are also a larger number of genes in strain 86-028NP: 1942 compared to 1743 in strain Rd. The gene complement was compared to that of strain Rd using the Seqman program in the DNASTAR suite. With 80% identity at the nucleotide level as a cutoff value, 285 ORFs were identified in the 86-028NP genome that were absent from the strain Rd genome and 167 ORFs were identified in the strain Rd genome that are absent from the strain 86-028NP genome.

Strain 86-028NP, like strain Rd, has six ribosomal operons. Using tRNAscan-SE v1.11, 58 tRNA genes were identified in the strain 86-028NP genome, representing the 20 common amino acids. The tRNA-Glu, tRNA-Ala and tRNA-Ile genes were located in spacer regions between the 16S and 23S ribosomal RNA genes. A tRNA gene containing the UCA anticodon was also identified. This anticodon corresponds to an opal stop codon and is typically associated with an opal-suppressing tRNA that incorporates selenocysteine. The tRNA is adjacent to two genes encoding selB (NTHI0836), a Sec tRNA specific elongation factor, and selA (NTHI0835), the enzyme that converts serine to dehydroalanine preparatory to forming selenocysteine by incorporation of selenium (Forchhammer et al., Nature 342:453-6, 1989) The selD gene (NTHI0297), encoding selenophosphate synthetase was also identified. The importance of this selenocysteine system is evidenced by the coding sequence for the alpha subunit of formate dehydrogenase (NTHI0007) containing an inframe TGA stop codon that is presumably read as a selenocysteine codon. The inframe TGA stop codon was previously noted in the current annotation of the strain Rd formate dehydrogenase gene (GenPept Accession P46448).

A gross comparison between the genomes involving analysis of the gene order of strain 86-028NP and that of strain Rd reveals a single major rearrangement in the form of a large inversion. This 471 kb inversion represents almost 25% of the strain 86-028NP genome and is bounded by NTHI1391, and NTHI1394 (homologues of HI1218 and HI1645 respectively) and by NTHI1949 and NTHI1950 (homologues of HI1219 and HI1647 respectively). HI1219 and HI1646 are partially duplicated genes in strain Rd annotated as cmkA and cmkB (cytidylate kinases). One cmk gene (NTHI1949) is present in strain 86-028NP with a small cmk-like fragment between NTHI1391 and NTHI1394. Several clones from the scaffolding library overlap each end of the inversion in the 86-028NP genome validating our assembly. Within this large inversion are several insertions, the largest of which are approximately 13 kb, 27 kb and 51 kb in size. These regions contain predominantly hypothetical and conserved hypothetical genes as well as a number of homologues of phage genes. For example, the 27 kb insertion contains remnants of HP1- and HP2-like phage genes. The largest insert is bounded by homologues of integrase genes. In strain Rd, a mu-like phage is localized to this region (Morgan et al., J Mol Biol 317:337-59, 2002) This phage is not present in the strain 86-028NP genome. Also within the large inverted region is a 21 kb inversion that restores synteny with the Rd genome.

In addition to the large inversion, strain 86-028NP has other regions of divergence from co-linearity with the strain Rd genome. These include 9 regions greater than 5 kb, which contain sequences with no apparent homology to DNA that is present in strain Rd. Two of these regions contain the HMW adhesins that are discussed below. Hypothetical genes predominate in six of the unique regions. The ninth region is approximately 56 kb in size. It lies between NTHI0100 and NTHI0165. BLASTn analysis indicated that genes in this region, designated ICEHin86-028NP, have high homology to genes in the H. influenzae type b plasmid, ICEHin1056 (Mohd-Zain et al., J Bacteriol 186:8114-22, 2004). ICEHin1056 is a member of an extended family of genomic islands that are defined by a series of common core genes (Mohd-Zain et al., J Bacteriol 186:8114-22, 2004). ICEHin86-028NP possesses homologues of 45 ICEHin1056 ORFs. These include ORFs near the 5′ end of ICEHin86-028NP, including the defined core genes, that primarily encode proteins with putative roles in plasmid replication and conjugation and ORFs near the 3′ end that primarily encode conserved hypothetical proteins with motifs that suggest that they may be either membrane associated or exported. Notably, ICEHin86-028NP lacks the genes encoding proteins involved in tetracycline, chloramphenicol and (3-lactam resistance found in ICEHin1056. Scattered within ICEHin86-028NP are a transposase, resolvases, and a putative integrase regulator suggesting that ICEHin86-028NP is a composite element derived from several mobile genetic elements.

ICEHin1506 has a sequence designated as an attP site 5′ the first gene. In strain 86-028NP, a perfect copy of this attP site is present 5′ to NTHI0101 and a copy of this attP site, with a single nucleotide change, is present 3′ of NTHI0164. The attP sites are implicated in the incorporation of mobile genetic elements into bacterial chromosomes to form genomic islands, possibly suggesting a mechanism by which this large section of genetic material became integrated into the strain 86-028NP genome (Dimopoulou et al., Antimicrob Agents Chemother 46:1602-3, 2002). ICEHin86-028NP has a G+C content of 39%, lower than any of the other related genomic islands and close to strain 86-028NP's overall genome G+C content of 38%. This implies a long-term genomic association for this element. The presence of this element with its complement of genes homologous to those in ICEHin1506 (Dimopoulou et al., Antimicrob Agents Chemother 46:1602-3, 2002) which are thought to encode membrane-associated and secreted proteins may have important implications for the virulence of strain 86-028NP.

Several members of the Pasteurellaceae including Haemophilus ducreyi, Pasteurella multocida and Actinobacillus actinomycetemcomitans produce well characterized protein toxins. In contrast, H. influenzae does not appear to produce protein toxins and genes encoding putative protein toxins were not identified in the strain 86-028NP genome. In H. influenzae, the genes encoding glycosyltransferases responsible for endotoxin biosynthesis and genes encoding proteins that give the bacteria enhanced “fitness” during the process of infection have generally been considered virulence determinants. These genes include those that encode adhesins, the heme and haemoglobin binding proteins as well as the genes that encode proteins that protect against oxidative stress.

Contingency Genes

H. influenzae has a limited number of two-component regulatory systems and other global regulators. Moxon and co-workers have argued that loci termed “simple contingency loci” provide an alternative mechanism for regulating gene expression, thus increasing the fitness of an organism by contributing to that organism's ability to rapidly respond to changing environmental conditions. These loci contain short tandem sequence repeats either within, or 5′ to, a coding region. During DNA replication, addition or loss of a repeat within a reading frame results in an alteration in the reading frame. When localized 5′ to a coding region, addition or loss of a repeat results in a change in promoter activity (Bayliss et al., Clin Invest 107:657-62, 2001). Loci containing simple sequence repeats have been studied extensively in H. influenzae, for example (Hood et al., Proc Natl Acad Sci USA 93:11121-5, 1996). Several of the loci described in the following sections as phase variable contain simple sequence repeats.

Adhesins

Strain 86-028NP possesses a number of genes which encode products that primarily function in adherence to host cells (Table 8). One of these, the outer membrane protein P5, has previously been identified and its function carefully dissected (Jiang et al., Infect Immun 67:187-92, 1999; Kennedy et al., Infect Immun 68:2756-65, 2000; Novotny et al., J Immunol 171:1978-83, 2003; Novotny et al., Infect Immun 68:2119-28, 2000; Novotny et al., Vaccine 20:3590-7, 2002; Sirakova et al., Infect Immun 62:2002-20, 1994). Strain 86-028NP possesses a gene cluster containing four genes that are homologues of pilABCD from strain Rd, Actinobacillus pleuropneumoniae and P. multocida (Bakaletz et al., Infect Immun 73:1635-4, 2005; Doughty et al., Vet Microbiol 72:79-90, 2000; Ruffolo et al., Infect Immun 65:339-43, 1997 Stevenson et al., Vet Microbiol 92:121-34, 2003). These genes together with the comE gene and genes yet to be identified encode a type IV pilus that has a role in adherence of strain 86-028NP to nasopharyngeal tissues (Kennedy et al., Infect. Immun., 68: 2756-2765, 2000).

Strain 86-028NP possesses two high molecular weight (HMW) adhesin gene clusters that are absent in strain Rd. The high molecular weight adhesins were first characterized in NTHi, strain 12, which has two HMW gene clusters, each encoding three proteins (HMWA, HMWB and HMWC). HMWA is the structural component of the adhesin, HMWB has a role in trans-membrane translocation, while HMWC is required for glycosylation of HMWA (Barenkamp et al., Infect Immun 60:1302-13, 1992;. Barenkamp et al., Infect Immun 62:3320-8; 1994; Grass et al., Mol Microbiol 48:737-51, 2003; St Geme et al., Mol Microbiol 27:617-30, 1998). Similarly, strain 86-028NP's two HMW gene clusters contain homologues of the hmwA, B and C genes in the same gene context as in strain 12 (Buscher et al., J Bacteriol 186:4209-17, 2004). The HMW1A and HMW2A proteins from strain 86-028NP are 72% identical, with the major area of divergence, including a 41 amino acid insertion in HMW2A, toward the C-termini. The paired HMWB and HMWC proteins from strain 86-028NP are 99% identical, respectively. The sequence ATCTTTC is repeated 17 times upstream of hmw1A and 23 times upstream of hmw2A. In strain 12, 16 repeats of this sequence are found 5′ of each hmw gene cluster (Barenkamp et al., Infect Immun 60:1302-13, 1992).

Hap is an autotransported protein with a domain homologous to the catalytic domain of IgA1 proteases. The NTHI0354 gene encodes a protein with 83% identity to Hap from the NTHi strain N187 (St Geme et al., Mol Microbiol 14:217-3, 1994). Strain 86-028NP, along with other NTHi strains that possess HMW1 and HMW2, lacks the gene encoding Hia, another Haemophilus adhesin (Barenkamp et al., Mol Microbiol 19:1215-23, 1996). Strain 86-028NP also lacks the hif gene cluster, encoding the hemagglutinating pilus as we previously reported (Munson et al., Infect Immun 72:3002-10, 2004).

TABLE 8 NTHi genes that encode proteins that primarily function in adherence to host cells Gene SEQ ID Contingency NTHI# HI# name NO: Function Repeats 354 hap 1080 Adhesion and penetration protein Hap 406 296 pilD 1125 Putative type 4 prepilin-like protein specific leader peptidase (EC 3.4.23.43) 407 297 pilC 1126 Putative type IV pilin secretion protein 408 298 pilB 1127 Putative type IV pilin secretion protein 409 299 pilA 1128 Type IV pilin subunit protein 1332 1164 ompP5 1953 Outer membrane protein P5 (OMP P5- homologous adhesin) 1448 hmw2C 2057 HMW2C, putative glycosyltransferase involved in glycosylation of HMW1A and HMW2A 1449 hmw2B 2058 HMW2B, OMP-85-like protein required for HMW1A and HMW2A secretion 1450 hmw2A 2059 HMW2A, high molecular weight adhesin 2 ATCTTTC repeated 23 times, 5′ of gene 1983 hmw1A 2530 HMW1A, high molecular weight adhesin 1 ATCTTTC repeated 17 times, 5′ of gene 1984 hmw1B 2531 HMW1B, OMP-85-like protein required for secretion of HMW1A and HMW2A 1985 Hmw1C 2532 HMW1C, putative gylcosyltransferase involved in glycosylation of HMW1A and HMW1B

In Tables 8, 9 and 10, the “NTHI number” refers to the locus tag number within the NTHi, strain 86-028NP genome as indicated at the Microbial-Pathogenesis H. influenzae 86028 NP web site and in Genbank Accession No. CP000057. The HI number” refers to the corresponding locus tag number in the TIGR (The Institute for genomic redearc

Lipooligosaccharide Synthesis [See [Page 13 of Original Application]

The structure, biosynthesis and role in virulence of H. influenzae lipooligosaccharide (LOS) has been studied extensively. Table 9 contains a list of genes involved in lipooligosaccharide biosynthesis. Strain 86-028NP has the full complement of genes required to synthesize the heptose-Kdo-Lipid A portion of LOS. The lgtF and lpsA genes encode glycosyltransferases that add glucose, and glucose or galactose, to heptose residues 1 and 3, respectively. Both of these genes are present in the strain 86-028NP genome, therefore it is likely that carbohydrate chains can be extended from the heptose 1 and heptose 3 residues of the strain 86-028NP LOS (Hood et al., Microbiology 150:2089-97, 2004). In the serotype b strain RM153, the lic2C gene encodes a glucosyltransferase that adds glucose to heptose 2 (Hood et al., Microbiology 150:2089-97, 2004). In the strain 86-028NP genome, this gene contains a frame shift. The phase variable lic2A and licA genes, encoding a galactosyltranferase and choline kinase, respectively, are present in the strain 86-028NP genome (High et al., Mol Microbiol 9:1275-82, 1993; Hood et al., Glycobiology 11:957-67, 2001; Weiser et al., Infect Immun 65:943-50, 1997). The lex2B gene which encodes a glucosyltransferase in the serotype b strain DL42, as well as a number of other serotypeable strains, is present in the strain 86-028NP genome (Griffin et al., Microbiology 149:3165-75, 2003; Jarosik et al., Infect Immun 62:4861-7, 1994). Five-prime to the lex2B gene in strain DL42 is the short phase variable lex2A gene. In strain 86-028NP, this gene is out-of-frame compared to the DL42 sequence (Genbank Accession U05670), due to the loss of one tetranucleotide repeat and a 5 bp deletion. Recently, Hood and co-workers described a locus in strain Rd, designated hmg, that contains HI0866 through HI0874 (Hood et al., J Bacteriol 186:7429-39, 2004). With the exception of a homologue of rmlB, these genes are absent from the strain 86-028NP genome. This includes the siaA gene which encodes a sialyltransferase recently shown to be important in biofilm formation in NTHI strain 2019 (Greiner et al., Infect Immun 72:4249-60, 2004; Jones et al., J Biol Chem 277:14598-611, 2002). Two copies of a homologue of the lic3A gene, encoding an alternative sialyltransferase, were identified in the strain 86-028NP genome (Hood et al., Mol Microbiol 39:341-50, 2001; Jones et al., J Biol Chem 277:14598-611, 2002), as well as a copy of the lsgB gene that encodes another sialyltransferase Jones et al., J Biol Chem 277:14598-611, 2002).

TABLE 9 NTHi genes involved in lipooligosaccharide biosynthesis Gene SEQ ID Contingency NTHI# HI# name NO: Function Repeats 68 58 kdsB 831 3-deoxy-D-manno-octulosonic acid cytidylyltransferase 69 59 lpxK 832 Tetraacyldisaccharide 4′-kinase 72 60 msbA 833 Lipid A export ATP-binding protein msbA 296 199 msbB 1029 Lipid A biosynthesis (KDO)2-(lauroyl)-lipid IVA acyltransferase 365 258 lgtC 1090 UDP-galactose--lipooligosaccharide GACA repeated 10 galactosyltransferase times, in frame 366 260 orfM 1091 Xanthosine triphosphate pyrophosphatase 367 260.1 kdkA 1092 3-deoxy-D-manno-octulosonic acid kinase 368 261 opsX 1093 ADP-heptose--lipooligosaccharide heptosyltransferase I 383 275 lpt6 1107 PE-tn-6--lipooligosaccharide phosphorylethanolamine transferase 471 351 galE 1184 UDP-glucose 4-epimerase 472 352 lic3A 1185 CMP-neu5Ac--lipooligosaccharide alpha 2-3 CAAT repeated 18 sialyltransferase times, in frame 512 391 Predicted acyltransferase AGCA repeated 8 times, in frame 649 523 waaQ 1350 ADP-heptose--lipooligosaccharide heptosyltransferase III 677 550 lic2A 1378 UDP-galactose--lipooligosaccharide CAAT repeated 14 galactosyltransferase times, in frame 772 652 kdtA 1462 3-deoxy-D-manno-octulosonic acid transferase 773 653 lgtF 1463 UDP-glucose--lipooligosaccharide glucosyltransferase 892 735 lpxH 1569 UDP-2,3-diacylglucosamine hydrolase 899 740 pgmB 1575 phosphoglucomutase 913 lex2B 1586 UDP-glucose--lipooligosaccharide glucosyltransferase 926 765 lpsA 1597 lipooligosaccharide glycosyltransferase 976 812 galU 1644 UTP--glucose-1-phosphate uridylyltransferase 1034 lic3A2 1696 CMP-neu5Ac--lipooligosaccharide alpha 2-3 CAAT repeated 18 sialyltransferase times, in frame 1037 873 rmlB 1698 dTDP-glucose 4,6-dehydratase 1082 915 lpxC 1739 UDP-3-O-[3-hydroxymyristoyl] glucosamine N-acyltransferase 1180 1005 Predicted PE--lipooligosaccharide phosphorylethanolamine transferase 1220 1060 lpxB 1858 Lipid-A-disaccharide synthase 1222 1061 lpxA 1859 Acyl-[acyl-carrier-protein]--UDP-N- acetylglucosamine O-acyltransferase 1224 1064 Predicted PE--lipooligosaccharide phosphorylethanolamine transferase 1272 1105 rfaF 1900 ADP-heptose--lipooligosaccharide heptosyltransferase II 1278 1114 rfaD 1906 ADP-L-glycero-D-manno-heptose-6- epimerase 1312 1144 gmbA 1934 UDP-3-O-[3-hydroxymyristoyl] N- acetylglucosamine deacetylase 1350 1181 gmhA 1969 Phosphoheptose isomerase 1474 1578 lgtD 2079 Putative UDP-glcNAc--lipooligosaccharide N- Acetylglucosamine glycosyltransferase 1576 1557 kdsA 2172 Phospho-2-dehydro-3-deoxyoctonate aldolase and 3-deoxy-D-manno-octulosonic acid 8-phosphate synthetase 1594 1540 licD 2189 Phosphorylcholine transferase 1595 1539 licC 2190 Protein licC, CTP--phosphocholine cytidylyltransferase 1596 1538 licB 2191 Protein licB, putative cho;ine uptake protein 1597 1537 licA 2192 Protein licA, choline kinase CAAT repeated 15 times, in frame 1606 1527 htrB 2200 Lipid A biosynthesis lauroyl acyltransferase 1607 1526 rfaE 2201 ADP-heptose synthase 1664 1337 mrsA 2251 Predicted phosphomannomutase 1750 Putative glycosyltransferase, glycosyl GACA repeated 14 transferase family 8 protein times, in frame 1769 Putative glycosyltransferase CCAA repeated 17 times, out of frame 1891 1279 siaB 2433 CMP-neu5Ac synthetase 1921 1244 Possible polysaccharide biosynthesis protein 2002 1695 lsgF 2548 Putative UDP-galactose--lipooligosaccharide galactosyltransferase 2003 1696 lsgE 2549 Putative UDP-galactose--lipooligosaccharide galactosyltransferase 2004 1697 lsgD 2550 Putative UDP-glcNAc--lipooligosaccharide N- Acetylglucosaminyl glycosyltransferase 2005 1698 lsgC 2551 Putative UDP-galactose--lipooligosaccharide galactosyltransferase 2006 1699 lsgB 2552 CMP-N-acetylneuraminate-beta- galactosamide-alpha-2,3-sialyltransferase 2007 1700 lsgA 2553 Putative lipooligosaccharide flippase 2025 1716 wecA 2569 Undecaprenyl-phosphate alpha-N- acetylglucosaminyl 1-phosphate transferase

Iron Acquisition

H. influenzae strains have an absolute requirement for either heme or iron, together with protophorphyrin IX (PPIX), the immediate precursor of heme (Evans et al., J Med Microbiol 7:359-65, 1974; White et al., J Bacteriol 85:842-50, 1963). Table 10 contains a list of genes involved in iron acquisition. Three haemoglobin and haemoglobin-haptoglobin binding proteins HgpA, HgpB and HgpC, were identified in H. influenzae type b, strain HI689 (Jin et al., Microbiology 145 (Pt 4):905-14, 1999; Morton et al., Infect Immun 67:2729-39, 1999; Ren et al., Infect Immun 66:4733-41, 1998). In strain HI689, these genes have CCAA tetranucleotide repeats and are known to be regulated by slip-strand mispairing. Two of these genes are present in strain 86-028NP. They both contain CCAA repeats; the hgpB gene is in-frame while the hgpC gene is out-of-frame. The derived amino acid sequence of a third gene that contains CCAA repeats is 45% identical to hgpA. We have designated this gene hgpD. This gene is out-of-frame. Homologues of the hxuABC genes of H. influenzae type b that encode heme and heme-hemopexin complexes (Cope et al., Infect Immun 69:2353-63, 2001; Cope et al., Infect Immun 66:4511-6, 1998; Cope et al., J Bacteriol 177:2644-53, 1995) as well as a homologue of the hemR receptor were identified. Strain 86-028NP also has the gene encoding the heme-binding lipoprotein HbpA (Heath et al., Pediatr Infect Dis J 20:300-5, 2001). Downstream of hbpA is NTHI1022, a hypothetical gene whose product is a member of COG0748, a cluster that includes putative heme utilization proteins. A homologue of the hup gene, recently identified in H. influenzae type b, that encodes a general heme utilization protein, was also identified (Morton et al., Microbiology 150:3923-33, 2004).

In addition to the heme transport systems, iron transport systems were also identified. The hitABC genes encode the FbpABC proteins respectively, members of a highly specific ferric iron ABC transport system that was elegantly characterized by complementing a siderophore-deficient E. coli strain with the hitABC genes cloned from an H. influenzae type b strain (Anderson et al., J Bacteriol 186:6220-9, 2004). Transferrin-binding proteins 1 and 2 encoded by tbpAB (Gray-Owen et al., Infect Immun 63:1201-10, 1995; Gray-Owen et al., Infect Immun 63:3809-15, 1995) as well as genes designated hfeABCD that are homologues of an ABC transport system involved in iron uptake, originally characterized in Yersinia pestis (Bearden et al., J Bacteriol 180:1135-47, 1998) were identified. This latter gene cluster is also present in strain Rd. NTHI2035 encodes a putative homologue of the NRAMP family of Mn²⁺ and Fe²⁺ transporters (Richer et al., J Mol Evol 57:363-7, 2003).

As noted above, H. influenzae can use iron, together with PPIX, as a source of heme for growth in vitro. The hemH gene encoding ferrochelatase, which catalyzes the incorporation of iron into PPIX (Schlor et al., Infect Immun 68:3007-9, 2000), was identified. The gene encoding the global regulator, Fur, was also identified (Andrews et al., FEMS Microbiol Rev 27:215-37, 2003; Smoot et al., J Med Microbiol 48:629-3, 1999).

TABLE 10 NTHi Genes Involved in Iron Acquisition Gene SEQ ID Contingency NTHI# HI# name NO: Function Repeats 177 97 hitA 931 hFbpA, Iron-utilization periplasmic protein 179 98 hitB 932 hFbpB, Iron(III)-transport system permease protein 180 99 hitC 933 hFbpC, Iron-utilization ATP-binding protein 202 113 hemR 951 Hemin receptor 284 190 fur 1020 Ferric uptake regulation protein 369 262 hxuC 1094 Heme/hemopexin-binding protein C (Heme: hemopexin utilization protein C) 370 263 hxuB 1095 Heme/hemopexin-binding protein B (Heme: hemopexin utilization protein B) 371 264 hxuA 1096 Heme/hemopexin-binding protein A (Heme: hemopexin utilization protein A) 477 359 hfeD 1090 Putative ABC-type chelated iron transport system, permease component 478 360 hfeC 1191 Putative ABC-type chelated iron transport system, permease component 479 361 hfeB 1192 Putative ABC-type chelated iron transport system, ATPase component 481 362 hfeA 1193 Putative periplasmic chelated iron binding protein 736 hgpD 1431 Hemoglobin-haptoglobin binding protein D CCAA repeated 17 (Hemoglobin-haptoglobin utilization protein times, out of frame D) 782 661 hgpB 1472 Hemoglobin-haptoglobin binding protein B CCAA repeated 12 (Hemoglobin-haptoglobin utilization protein B) times, in frame 840 712 hgpC 1523 Hemoglobin-haptoglobin binding protein C CCAA repeated 20 (Hemoglobin-haptoglobin utilization protein times, out of frame C) 1021 853 hbpA 1684 Heme-binding protein A (Hemin-binding lipoprotein) 1168 994 tbp1 1817 Transferrin-binding protein 1 1169 995 tbp2 1818 Transferrin-binding protein 2 1329 1160 hemH 1950 Ferrochelatase 1390 1217 hup 2004 Heme utilization protein 2035 1728 2577 Mn2+ and Fe2+ transporter of the NRAMP family

Oxidative Stress

Although necessary for growth, the active acquisition of iron can have deleterious effects on bacterial cells. Through the Fenton reaction, iron can react with hydrogen peroxide and generate highly reactive hydroxyl radicals. These products have profound effects, including lipid peroxidation and damage to both iron-containing enzymes and DNA (Imlay, Annu Rev Microbiol 57:395-418, 2003). The best-known defense system against hydroxyl radicals consists of superoxide dismutase A and B which convert highly reactive superoxide to hydrogen peroxide, which is then converted, by catalase, into water and oxygen (Demple, Annu Rev Genet. 25:315-37, 1991). Strains 86-028NP and Rd contain the sodA gene (NTHI1251), but lack the sodB gene. Both strains also possess a catalase gene hktE (NTHI1099) (Bishai et al., J Bacteriol 176:2914-21, 1994), the oxyR gene (NTHI0704) encoding a primary regulator of genes involved in protection against oxidative stress (Maciver & Hansen, Infect Immun 64:4618-29, 1996; Pomposiello et al., Trends Biotechnol 19:109-14, 2001) and the gene encoding a chimeric peroxidase termed Prx/Grx that has a glutathione-dependent role in protection against small alkyl hydroperoxides (Pauwels et al., J Biol Chem 278:16658-66, 2003; Vergauwen et al., J Bacteriol 185:5555-62, 2003; Vergauwen et al., J Bacteriol 185:1572-81, 2003). We previously identified NTHI0212, a gene encoding a homologue of the P. multocida peroxiredoxin, TsaA that is absent in strain Rd (Munson et al., Infect Immun 72:3002-10, 2004). Strain 86-028NP, however, lacks AhpF, a dedicated alkyl hydroperoxide reductase known to be involved in the reduction of TsaA in Salmonella (Poole et al., Biochemistry 39:6602-15, 2000). Further protection against oxidative stress may be afforded by the ferritin-like proteins encoded by the ftnA and ftnB (NTHI1773 and NTHI1772, respectively) genes. Over-expression of these proteins were shown to protect an iron overloaded E. coli fur mutant against oxidative damage Touati et al., J Bacteriol 177:2305-14, 1995). A conserved hypothetical gene, NTHI1817, encodes a protein with homology to a DNA-binding ferritin-like protein. This is a member of the Dps family of non-specific DNA binding proteins, which in S. enterica have roles in protection against oxidative stress, both in the presence of iron and during phagocytosis, and are important for virulence in a murine model of Salmonella infection (Halsey et al., Infect Immun 72:1155-8, 2004) In E. coli, Dps was shown to preferentially bind iron that had been oxidized by hydrogen peroxide, thus having an important role in abrogating the production of hydroxyl radicals generated via the Fenton reaction (Zhao et al., J Biol Chem 277:27689-96, 2002).

Secretion

In addition to the Sec system, strain 86-028NP has genes that encode the TatA, B and C proteins, cytoplasmic membrane-associated proteins that are involved in a Sec-independent transport of proteins with twin arginines in their signal peptides (NTHI0279, NTHI0280 and NTHI0282) (Bolhuis et al., J Biol Chem 276:20213-9, 2001; Yen et al., Arch Microbiol 177:441-50, 2002). As previously reported, strain 86-028NP possesses NTHI0585, the gene encoding the autotransported protein Lav (Munson et al., Infect Immun 72:3002-10, 2004). This protein is absent in strain Rd, present in Neisseria and appears, within Haemophilus, to be restricted to pathogenic strains (Davis et al., J Bacteriol 183:4626-35, 2001). Strain 86-028NP also has the gene encoding an IgA protease (NTHI1164) (Poulsen et al J Bacteriol 174:2913-21, 1992), and as noted above, the gene encoding the Hap adhesin. Both are proteins of the autotransporter class. As described above, the HMW adhesins are members of the two-partner secretion pathway group of proteins.

Outer Membrane Proteins

A number of outer membrane protein (OMP) encoding genes have been identified by homology to those in other Haemophilus isolates. These include the major OMPs that were all originally identified in H. influenzae type b; the surface expressed P1 (NTHI0522), the porin P2 (NTHI0225), the phosphomonoesterase and heme transporter P4 (NTHI0816), the adhesin P5 (NTHI1332) and the lipoprotein P6 (NTHI0501). Strain 86-028NP also shares a number of minor OMPs with other Haemophilus strains. These include D15 and the transferrin binding proteins from H. influenzae type b, as well as a homologue of OMP26, which was identified in NTHi strain 289 (Munson et al., Infect Immun 56:2235-42, 1988; Munson et al., Infect Immun 49:544-9, 1985; Munson et al., J Clin Invest 72:677-84, 1983; Reidl et al., J Exp Med 183:621-9, Reilly et al., J Bacteriol 181:6797-805, 1999; Reilly et al., FEBS Lett 494:19-23, 2001). All have subsequently been characterized in NTHi strains and analyzed as potential vaccine candidates (Poolman et al., Vaccine 19 Suppl 1:S108-15, 2000; Murphy et al. Curr Opin Infect Dis 16:129-34, 2003; McMichael et al., Curr Opin Investig Drugs 4:953-8, 2003 Cripps et al. Immunol Cell Biol 81:46-51, 2003; Bakaletz et al. Ann Otol Rhinol Laryngol Suppl 188:82-94, 2002).

Restriction Enzymes Systems:

Strain 86-028NP lacks the HindIII and HindIII type II restriction systems (Fleischmann et al., Science 269:496-512. 80, 1995; Nwankwo et al., Gene 150:75-80. 104, 1994, Smith, & Marley. Methods Enzymol 65:104-8, 1980). In contrast, genes encoding the HaeII system that was originally identified in H. aegyptius (Slatko et al., Gene 74:45-50, 1988) are present in the strain 86-028NP genome but absent in strain Rd. Both strain 86-028NP and strain Rd have Hsd type I restriction systems encoding a methylransferase (HsdM), a sequence recognition protein (HsdS) and a restriction enzyme (HsdR) (Roberts et al., Nucleic Acids Res 31:1805-12, 2003). These genes are adjacent in the strain Rd genome (HI1285-HI1287). The 86-028NP genome contains 3 hsd-like loci that each contain 4 genes. One hsd system is encoded by NTHI1838-NTHI1843. In this gene cluster, NTHI1841 encodes a hypothetical protein. A second hsd-like locus is encoded by NTHI0314-NTHI0318. In this gene cluster, NTHI0316 encodes a putative anticodon nuclease. This hsd-like system may be similar to the prr system in E. coli (Tyndall et al., J Mol Biol 237:266-74, 1994). A third hsd-locus is encoded by NTHI0188-NTHI0193. In this gene cluster, NTHI0190 encodes a predicted transcriptional regulator with a helix-turn-helix domain. 

1. An isolated polynucleotide comprising the nucleotide sequence of any one of SEQ ID NOS:577 to 579, SEQ ID NOS: 589 to 614, SEQ ID NOS: 675 to 685, SEQ ID NO: 615, SEQ ID NO: 617, SEQ ID NO: 619, SEQ ID NO: 621, SEQ ID NO: 623, SEQ ID NO: 625, SEQ ID NO: 627, SEQ ID NO: 629, SEQ ID NO: 631, SEQ ID NO: 633, SEQ ID NO: 635, SEQ ID NO: 637, SEQ ID NO: 639, SEQ ID NO: 641, SEQ ID NO: 643, SEQ ID NO: 645, SEQ ID NO: 647, SEQ ID NO: 649, SEQ ID NO: 651, SEQ ID NO: 653, SEQ ID NO: 655, SEQ ID NO: 657, SEQ ID NO: 659, SEQ ID NO: 661, SEQ ID NO: 663, SEQ ID NO: 665, SEQ ID NO: 667, SEQ ID NO: 669, 671, SEQ ID NO: 673, SEQ ID NO: 686, SEQ ID NO: 688, SEQ ID NO: 692, SEQ ID NO: 694, SEQ ID NO: 696, SEQ ID NO: 698, SEQ ID NO: 700, SEQ ID NO: 702, SEQ ID NO: 704, SEQ ID NO: 706, SEQ ID NO: 708, SEQ ID NO: 710, SEQ ID NO: 712, SEQ ID NO: 714, SEQ ID NO: 716, SEQ ID NO: 718, SEQ ID NO: 720, SEQ ID NO: 722, SEQ ID NO: 724, SEQ ID NO:726, SEQ ID NO: 728, SEQ ID NO: 730, SEQ ID NO: 732, SEQ ID NO: 734, SEQ ID NO: 736, SEQ ID NO: 738, SEQ ID NO: 740, SEQ ID NO: 742, SEQ ID NO: 744, SEQ ID NO: 746, SEQ ID NO: 748, SEQ ID NO: 750, SEQ ID NO: 752, SEQ ID NO: 754, SEQ ID NO: 756, SEQ ID NO:758, SEQ ID NO:760, SEQ ID NO: 762, SEQ ID NO: 764, SEQ ID NO: 766, SEQ ID NO:768 SEQ ID NO: 770 or SEQ ID NOS: 773 to
 2593. 2. An isolated polypeptide comprising an amino acid sequence encoded by a nucleotide sequence set of claim 1 or a fragment thereof.
 3. An isolated polypeptide comprising an amino acid sequence of any one of SEQ ID NO: 616, SEQ ID NO: 618, SEQ ID NO: 620, SEQ ID NO: 622, SEQ ID NO: 624, SEQ ID NO: 626, SEQ ID NO: 628, SEQ ID NO: 628, SEQ ID NO: 630, SEQ ID NO: 632, SEQ ID NO: 634, SEQ ID NO: 636, SEQ ID NO: 638, SEQ ID NO: 640, SEQ ID NO: 642, SEQ ID NO: 644, SEQ ID NO: 646, SEQ ID NO: 648, SEQ ID NO: 650, SEQ ID NO: 652, SEQ ID NO: 654, SEQ ID NO: 656, SEQ ID NO: 658, SEQ ID NO: 660, SEQ ID NO: 662, SEQ ID NO: 664, SEQ ID NO: 666, SEQ ID NO: 668, SEQ ID NO: 670, SEQ ID NO: 672, SEQ ID NO: 674, SEQ ID NO: 687, SEQ ID NO: 689, SEQ ID NO: 691, SEQ ID NO: 693, SEQ ID NO: 695, SEQ ID NO: 697, SEQ ID NO: 699, SEQ ID NO: 701, SEQ ID NO: 703, SEQ ID NO: 705, SEQ ID NO: 707, SEQ ID NO: 709, SEQ ID NO: 711, SEQ ID NO: 713, SEQ ID NO:715, SEQ ID NO: 717, SEQ ID NO: 719, SEQ ID NO: 721, SEQ ID NO:723, SEQ ID NO:725, SEQ ID NO:727, SEQ ID NO:729, SEQ ID NO: 731, SEQ ID NO: 733, SEQ ID NO: 735, SEQ ID NO: 737, SEQ ID NO: 739, SEQ ID NO: 741, SEQ ID NO: 743, SEQ ID NO: 745, SEQ ID NO: 747, SEQ ID NO: 749, SEQ ID NO: 751, SEQ ID NO: 753, SEQ ID NO: 755, SEQ ID NO: 757, SEQ ID NO: 759, SEQ ID NO: 761, 763, SEQ ID NO: 765, SEQ ID NO: 767, SEQ ID NO: 769, SEQ ID NO: 771 or SEQ ID NOS: 2581 to
 4414. 4. A composition comprising a polypeptide of claim 3 and a pharmaceutically acceptable carrier.
 5. An antibody that specifically binds to a polypeptide of claim 3 or fragment thereof.
 6. A composition comprising an antibody of claim 5 and a pharmaceutically acceptable carrier.
 7. A method for detecting NTHi bacteria in a biological sample comprising (a) contacting a polynucleotide of claim 1 or a fragment thereof with a biological sample, and (b) detecting hybridization of the polynucleotide within the sample.
 8. A method for detecting NTHi bacteria in a biological sample comprising: (a) contacting an antibody of claim 5 with a biological sample, and (b) detecting binding of the antibody within the sample.
 9. The method of claim 7 wherein the biological sample is selected from the group consisting of serum, sputum, ear fluid, blood, urine, lymphatic fluid, and cerebrospinal fluid.
 10. A method for eliciting an immune response to NTHi bacteria comprising administering an immunogenically effective dose of a polypeptide of claim 3 or a fragment thereof to a patient at risk of NTHi bacterial infection.
 11. A vaccine comprising a polypeptide of claim 3 or a fragment thereof and a pharmaceutically suitable carrier.
 12. The method of claim 10 wherein the immune response includes one or more of killing NTHi bacteria, slowing replication of NTHi bacteria or blocking binding of NTHi bacteria.
 13. The method of claim 10 wherein the NTHi infection is in the middle ear.
 14. A method of treating or preventing NTHi bacterial infection comprising administering a molecule that inhibits expression or activity of a polypeptide of claim 3 to an patient in need.
 15. The method of claim 14 wherein the NTHi polypeptide comprises the is encoded by an NTHi gene selected from the group consisting of hisB, lppB, sapA, rbsC, pure, rib, arcB, uxuA, licC, ispZ, mukF, glpR, ihfB, cspD, lay, HI1647, HI0094, HI1163, HI0665, HI1292, HI1064, HI1386, HI1462, HI1369, and HI1598.
 16. The method of claim 14 wherein the NTHi polypeptide comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 616, SEQ ID NO: 618, SEQ ID NO: 620, SEQ ID NO: 622, SEQ ID NO: 624, SEQ ID NO: 626, SEQ ID NO: 628, SEQ ID NO: 634, SEQ ID NO: 638, SEQ ID NO: 642, SEQ ID NO: 644, SEQ ID NO: 646, SEQ ID NO: 650, SEQ ID NO: 652, SEQ ID NO: 656, SEQ ID NO: 658, SEQ ID NO: 660, SEQ ID NO: 662, SEQ ID NO: 664, SEQ ID NO: 666, SEQ ID NO: 668, SEQ ID NO: 670, SEQ ID NO:672 and SEQ ID NO:
 674. 17. The method of claim 14 wherein the molecule administered to the patient in need is an antisense oligonucleotide.
 18. The method of claim 14 wherein the molecule administered to the patient in need is an antibody.
 19. The method of claim 14 wherein the molecule administered to the patient in need is a small molecule.
 20. The method of claims 14 wherein the NTHi infection is in the middle ear. 